poster abstracts

Transcription

poster abstracts

PROTEIN SCIENCE
A P U B L I C AT I O N O F T H E P R O T E I N S O C I E T Y
w w w. p ro t e i n s c i e n c e . o rg
Vol 23 Suppl 1 July 2014
The 28th Annual Symposium of The Protein Society
July 27–30, 2014 • San Diego, CA
PROGRAM & ABSTRACTS
Lalima G. Ahuja, Ph.D.
University of California, San Diego
Abstract: (POST 10-67) Tweaking the Spines of Kinase Structures
JULY 22-25, 2015
BARCELONA
The 29th Annual Symposium of The Protein Society
P RESIDENT ’ S W ELCOME
Dear Attendees,
As President of the Protein Society, it is my privilege and pleasure to
welcome you to the 28th Annual Symposium of The Protein Society. The
2014 Program Planning Committee has organized a terrific program with
a diverse array of topics and an exciting slate of speakers. The Program
opens with a plenary session featuring a talk by James H. Hurley
(University of California - Berkeley), the winner of the 2014 Hans Neurath
Award, and will conclude with a plenary session featuring a talk by 2014
Stein & Moore Award winner Nikolaus Pfanner (University of Freiberg).
Tuesday afternoon the Plenary Awards Session will include the
presentation of our other prestigious awards, the announcement of the
Best Poster Prize winners, and be followed by our Members Reception -I
hope to see you all there!
James U Bowie, PhD
President, The Protein Society
In addition to the great scientific sessions, the meeting offers many other opportunities for
learning and making new connections. Posters will be up for the entire meeting, which we hope
will facilitate interactions and select posters will receive Best Poster Awards. The Mentoring
Committee will be leading workshops on public speaking, scientific writing, and a workshop
called “How to Navigate Your Career” that will feature working scientists sharing advice on
topics like preparing your CV, online job-hunting, and salary negotiations. The Education
Committee is leading the Undergraduate Research Session, where outstanding undergraduate
researchers will present their work, and a luncheon for educators entitled “Assessing Student
Understanding of Foundational Concepts of Protein Structure and Function”. On Monday
night, both committees combine for The Education & Mentoring Committees’ Mixer -an
informal and fun opportunity to meet the committee members, make new connections, and find
out how you can get involved. Please consult your schedule for details about these and other
learning and networking opportunities, including an iPad giveaway, a chance for students and
postdocs to have dinner with a speaker or society leader of their choice, a protein WikiPedia
workshop, The Members Business meeting and more.
At the conclusion of the meeting you will receive an email invitation to participate in a survey,
and I hope you will take a moment to provide your perspective. The Society is always interested
in ways to more effectively achieve our mission, and your feedback will help us to do just that.
The Protein Society does not begin and end with the annual meeting. If you have creative ideas
for protein-focused gatherings throughout the year, we’d like to help, so check out our Minigrant
Program. Our journal, Protein Science, continues to go strong, so submit your work to Protein
Science and experience the pleasure of having manuscripts handled rapidly and fairly by real
scientists. Each year the first author of the top papers published in Protein Science are selected
to present their work at the Annual Symposium—a great opportunity for young investigators.
Follow us on Twitter and Facebook.
Thank you for being a part of the Protein Society’s 28th Annual Symposium.
Best Wishes,
James U. Bowie, PhD
Protein Society President
UCLA
T HANK Y OU
TABLE OF CONTENTS
Symposium Overview .................................................................................................... 3
Executive Council & Leadership ................................................................................. 4
Committees ...................................................................................................................... 5
Protein Society Awards ................................................................................................. 6
Call for 2015 Nominations ............................................................................................... 8
Hotel Floorplan ............................................................................................................... 9
General Information.................................................................................................... 10
Travel Awards ................................................................................................................ 12
Donor Acknowledgements ......................................................................................... 13
Daily Program ............................................................................................................... 14
Day 1 ................................................................................................................................14
Day 2................................................................................................................................ 17
Day 3................................................................................................................................21
Day 4............................................................................................................................... 24
Poster Sessions............................................................................................................. 26
Exhibitors ....................................................................................................................... 47
Exhibitor Workshops ..................................................................................................54
Speaker Abstracts ......................................................................................................... 55
Poster Abstracts ............................................................................................................ 71
Author Index ............................................................................................................... 274
T HANK Y OU
THE 2014 PROGRAM PLANNING COMMITTEE
Todd O. Yeates, Ph.D.
Professor, Department of Chemistry and
Biochemistry
University of California, Los Angeles
Jennifer R. Potts, Ph.D.
Professor and Chair of Molecular
Biophysics
Department of Biology
The University of York
Tarun Kapoor, Ph.D.
Pels Family Professor
Laboratory of Chemistry and Cell Biology
The Rockefeller University
CORPORATE SUPPORT
The Protein Society is grateful to the following corporate partnerns for
supporting the 28th Annual Symposium & The Protein Society:
28TH ANNUAL SYMPOSIUM SPONSORS
Proteins in Disease & Therapeutics
Research Session
Best Poster Competition
Complimentary Tote Bags
2014 CORPORATE MEMBERS
Gold Level
Bronze Level
SYMPOSIUM OVERVIEW
DAY 1
SUNDAY JULY 27TH
DAY 2
MONDAY JULY 28TH
Members Business
Meeting/New Member
Welcome Breakfast
7:00 AM
DAY 3
TUESDAY JULY 29TH
DAY 4
WEDNESDAY JULY 30TH
(75 min) - Marina Room
8:30 AM Opening Plenary Session
Protein Proteins in Protein Proteins In Closing Plenary Session &
Evolution Disease & Engineering Dynamic &
2014 Stein & Moore
Grand HAll C
Thera- & Synthetic Driven
Award Winner Talk
Grand Hall C & D
peutics
Biology
Processes
Grand Hall C & D
Grand
HAll
D
Grand
HAll
C
Grand
HAll
D
Protein Membrane
9:40 AM Translation Bacterial
& Folding Interactions
DegraProteins &
Grand HAll C Grand Hall D
dation
Receptors
& 2014 Hans Neurath
Award Winner Talk
Grand HAll C
11:30 -Exhibits & Poster Displays
(2 hrs) - Grand Hall A & B
11:30 AM
LUNCH
(2 hrs)
Grand HAll D
Exhibitor Workshop
Exhibitor Workshop
Exhibitor Workshop
NanoTemper
GE Healthcare
Technologies
EW I-47 NEW Amersham™ 600
EW III-49 Multimodal
Imager series (CCD-based Some like it hot: Determination
chromatography: A new
of Biomolecular Interactions generation of high selectivity
camera systems)
using MicroScale
(1 hr) - Gaslamp A
media for challenging
Thermophoresis
purifications
(1 hr) - Gaslamp A
(1 hr) - Gaslamp A
All Lunch Workshops from Noon - 1PM
GE Healthcare
Workshop: Undergrad Educator’s Workshop: Workshop: Workshop:
How to
Research Luncheon How to Write WikiPedia for
How to
(1 hr)
Improve Your Session
an Effective
Protein
Navigate
(1 hr)
Writing
(1 hr)
Bankers Hill
Gaslamp C & D
1:30 PM
Bankers Hill
End of Program
Paper
Scientists Your Career
(1 hr)
(1 hr)
(1 hr)
Gaslamp C & D Bankers Hill Gaslamp C & D
Frontier Proteins in Chemical
Cellular Plenary Awards Session
HighAltered
Biology & Structures
1:30 PM - 5:50 PM
Throughput States Enzymology Grand HAll D
Grand Hall C & D
Techniques Grand HAll D Grand HAll C
Grand HAll C
4:30 PM Exhibits & Poster Displays (2 hrs) - Grand Hall A & B
7:00 PM Networking Dinners (3hrs)
- Locations Vary
8:00 PM
Members Reception
Education & Mentoring
Committees’ Mixer
(3hrs) - Please View the
Program Addendum for
Location
(3 hrs)
Kin Spa Pool Deck, 3rd Floor
Following the Symposium, please take a few moments to share your feedback when
you receive the Attendee Survey
3
EXECUTIVE COUNCIL & LEADERSHIP
President
Secretary/Treasurer
James Bowie, University of
California, Los Angeles
Jacquelyn Fetrow, University of
Richmond
Past-President
Lynne Regan, Yale University
Lars Baltzer, Uppsala
University
Paula Booth, University
of Bristol
Charles Brooks, III,
University of Michigan
Johannes Buchner,
Technische Universität
München
Zengyi Chang, Peking
University
Stewart Fisher, SL
Fisher Consulting
Amy Keating,
Massachusetts Institute
of Technology
Jacqui Matthews,
University of Sydney
Haruki Nakamura,
Osaka University
Andreas Plückthun,
Universität Zurich
Catherine Royer,
Rensselaer Polytechnic
Institute
Michael Summers,
University of Maryland,
Baltimore County
Sarah Teichmann,
EMBL-EBI/Wellcome Trust
Sanger Institute
Brian Matthews (ex officio),
Editor in Chief, University of
Oregon
Jody McGinness (ex officio),
Executive Director, The Protein
Society
4
NOMINATING COMMITTEE
ABSTRACT REVIEW COMMITTEE
Mary Munson, University of Massachusetts
Medical School (Chair)
John Osterhout, Angelo State University (Chair)
Judith Frydman, Stanford University
Christopher Bystroff, Rensselaer Polytechnic
Institute
Matthais Mann, Max-Planck-Institut für Biochemie
Cory L Brooks, California State University - Fresno
Thomas Magliery, The Ohio State University
Andrew Miranker, Yale University
Paola Di Lello, Genentech, Inc.
Mary Munson, University of Massachusetts
Medical School
Francisco J. Enguita, University of Lisbon
Brenda Schulman, St. Jude Children's Research
Hospital
Wei Yang, LMB, NIDDK, NIH
John J. Dwyer, Ferring Research Institute
Angelo M. Figueiredo, University of East Anglia
Azucena G. Horta, Universidad Autonoma de
Nuevo Leon
Mary Konkle, Eastern Illinois Univerrsity
PUBLICATIONS COMMITTEE
Suresh Kumar, University of Arkansas
Amy Keating, Massachusetts Institute of
Technology (Chair)
Patricia Ann Mabrouk, Northeastern University
James Bowie, University of California - Los Angeles
Matthew Torres, Georgia Institute of Technology
Arthur Palmer, III, Columbia University
Lynne Regan, Yale University
Sudha Veeraraghavan, University of Maryland
School of Pharmacy
Jacquelyn Fetrow, University of Richmond
Bo Zhang, DuPont Industrial Biosciences
Brian Matthews, University of Oregon
Zhiwen Zhang, Santa Clara University
Todd Naumann, USDA-ARS-NCAUR
MENTORING COMMITTEE
Blake Hill, Medical College of Wisconsin
Judith Klein-Seetharaman, University of Pittsburgh
School of Medicine
Kevin Lumb, Merck & Company
Giovanna Ghirlanda, Arizona State University
Matthew Cordes, University of Arizona
Janel McLean, Vanderbilt University Medical
Center
Andrew Vendel, Eli Lilly and Company (Chair)
Patricia Jennings, University of California, San
Diego
Olve Peersen, Colorado State University
Stephen Fuchs, Tufts University
Carly Huitema, SIAF institute
Eric Sundberg, University of Maryland School of
Medicine
EDUCATION COMMITTEE
Ellis Bell, University of Richmond (Chair)
John Osterhout, Angelo State University
Margaret Cheung, University of Houston
Jane Richardson, Duke University
Matthew Gage, Northern Arizona University
Srebrenka Robic, Agnes Scott College
Lisa Gentile, University of Richmond
Peter Kahn, Rutgers University
Michael Summers, University of Maryland Baltimore County/HHMI
Betsy Komives, University of California - San Diego
Frieda Texter, Albright College
Mary Konkle, Eastern Illinois University
Jeff Watson, Gonzaga University
5
2014 PROTEIN SOCIETY AWARDS
The Carl Brändén Award, sponsored by Rigaku Corporation, is given to an
outstanding protein scientist who has also made exceptional contributions in
the areas of education and/or service to the science. The 2014 recipient of this
award is Dr. Stephen H. White (University of California, Irvine) in
recognition of his many contributions to the field of membrane protein
folding, his long service to the protein science community, and his skills as an
educator of graduate students and postdocs in particular. His research has
provided much fundamental insight into the thermodynamics of folding in and on the
membrane. Dr. White has made major contributions to multiple scientific societies, including
The Protein Society, and --among other service activities-- maintains the widely-utilized
Membrane Proteins of Known 3D Structure website.
Talk Title: Membrane Protein Folding: Biology Meets Thermodynamics
When: 5:20 PM Tuesday, July 29th
Where: Plenary Awards Session, 1st Floor, Grand Hall C & D
The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is
granted in recognition of exceptional contributions in protein science which
profoundly influence our understanding of biology. The 2014 award will be
presented to Dr. Judith Frydman (Stanford University) for exceptional
contributions to our understanding of protein folding in eukaryotic cells. Dr.
Frydman uses creative combinations of biochemical, genetic, biophysical, and
computational analysis to dissect the mechanisms by which cells manage
misfolded proteins, with a particular focus on the group II cytosolic chaperonin TRiC.
Talk Title: Chaperone Control of Cellular Proteostasis in Health and Disease
The Hans Neurath Award, sponsored by The Neurath Foundation,
recognizes individuals who have made a recent contribution of unusual merit
to basic research in the field of protein science. In 2014 the Hans Neurath
Awardee is Dr. James H. Hurley (University of California, Berkeley) for his
ground-breaking contributions to structural membrane biology and
membrane trafficking. Throughout his career, Dr. Hurley has produced
fundamental insights about proteins that function at the membrane-solvent
interface, including how signaling molecules interface with and are regulated by lipids,
mechanisms of vesicle formation by ESCRT machinery, and, most recently, the regulation of
clathrin coated vesicle formation and the initiation of autophagy.
Talk Title: The Coreography of Self-Cannibalism
When: 8:30 AM Sunday, July 27th
Where: Opening Plenary Session, 1st Floor, Grand Hall C & D
The Christian B. Anfinsen Award, sponsored by The Protein Society,
recognizes significant technical achievements in the field of protein science.
The recipient of this award in 2014 is Dr. Robert Tycko (National Institutes
of Health) for his seminal contributions to the development of modern solid
state nuclear magnetic resonance methods for structural investigations of
proteins and influential studies on amyoloid fibrils associated with
neurodegeneration and disease. Dr. Tycko’s achievements in this area are
highlighted by his recent presentation of the first detailed structure of β-amyloid fibrils from
Alzheimer's disease brain tissue and evidence for structural variations that underlie prion
diseases.
6
Talk Title: The Molecular Structural Basis for Amyloid Formation, in vitro and in vivo
The Emil Thomas Kaiser Award, sponsored The Protein Society,
recognizes a recent, highly significant contribution to the application of
chemistry in the study of proteins. The 2014 awardee is Dr. Carol Fierke
(Dept. of Chemistry, University of Michigan). Dr. Fierke is being recognized
for her exceptional contributions to our understanding of the metal
homeostasis, and to understanding of the structure and mechanism of
ribonuclease P. Her research has combined metallochemistry, biochemistry,
genetics and a wide range of spectroscopic techniques to uncover details of metal affinity,
substrate specificity, and roles to catalytic activity in numerous biologically and medically
important enzymes. Amongst her groundbreaking achievements include the development of
protein-based metal biosensors and techniques for monitoring metal ions in vivo for probing
metal transport and toxicity.
Talk Title: Histone Deacetylases: Substrate Selectivity and Regulation
The Protein Science Young Investigator Award, named for the
academic journal of the Society, recognizes an important contribution to the
study of proteins by a scientist still in the early stages of an independent
career. The 2014 awardee is Dr. M. Madan Babu (MRC Laboratory of
Molecular Biology, Cambridge UK). Dr. Babu is being recognized for his
contributions to our understanding of the principles of regulation in biological
systems – especially in the fields of intrinsically disordered proteins and gene
regulation. He was appointed in 2006 as one of the youngest independent group leaders in the
Structural Studies division at the LMB. Dr. Babu employs an interdisciplinary approach,
combining computational and experimental methods to investigate regulatory processes at
multiple scales of complexity (ranging from the molecular to the genome level) across a wide
range of model organisms.
Talk Title: Please consult the Program Addendum
The Stein & Moore Award, sponsored by The Protein Society, is named for
Nobel laureates Dr. William Stein and Dr. Stanford Moore. The award
venerates their contribution to understanding the connection between
chemical structure and catalytic activity of the active center of the
ribonuclease molecule. The 2014 awardee is Dr. Nikolaus Pfanner
(Institute for Biochemistry and Molecular Biology, University of Freiburg) in
recognition of his contributions to understanding pathways of cellular sorting
that underlie the biogenesis of mitochondria. His pioneering research includes the first
comprehensive proteomic analysis of mitochondria, the discovery of three new pathways of
mitochondrial import, and the reconstitution of mitochondrial import channels and unraveling
mechanisms of protein translocation.
Talk Title: Mitochondrial machinery for transport and assembly of proteins
When: 11:00 AM Wednesday, July 30th
Where: Closing Plenary Session, 1st Floor, Grand Hall C & D
7
CALL FOR NOMINATIONS
for the
2015 Awards of The Protein Society
Presented annually to distinguished scientists, the Protein Society Awards recognize excellence and
outstanding achievements in the multidisciplinary fields of protein science and honor distinguished
contributions in the areas of leadership, education, and service. The 2015 awards will be
presented at the 29th Annual Symposium of The Protein Society (July 22-25,2015, in
Barcelona, Spain). The deadline for submitting complete award nomination packages for the
2015 Awards cycle will be noon (EDT) on September 22, 2014
Note: the ability to submit a nomination is a benefit of membership.
8
HQ HOTEL
MANCHESTER GRAND HYATT, SAN DIEGO
LOBBY LEVEL
1 MARKET PLACE | SAN DIEGO,CA 92101, USA
2ND LEVEL, SEAPORT TOWER
GENERAL INFORMATION
Scientific Registration – Grand Hall
Foyer
The Registration Area will open at 4:00 PM on
Saturday, July 26th (refer to hours below).
Registration includes admission to all scientific
and poster sessions, exhibits, one copy of the
Program/Abstract book and one tote bag.
Registration does not include any meals.
Hours
Saturday 4:00 pm – 7:00 pm
Sunday 7:30 am – 5:00 pm
Monday 7:00 am – 5:00 pm
Tuesday 7:30 am – 5:00 pm
Registration Fees
Full Member Registration $400
Emeritus Member Registration $350
Early-Career Member Registration $350
Lab Staff Member Registration $350
Graduate Student Member Registration $225
Undergrad Member Registration FREE
Undergrad Non-Member Registration $25
Regular Non-Member Registration $700
Single-day Registration $400
Guest Registration (Networking Events) $95
Press Registration
Complimentary Press badges will be issued on-site to members of the working press and
freelance writers. Press credentials or an official letter from an editor bearing a letter of
assignment must be provided to register as press. Representatives of allied fields (public
relations, public information, public affairs) are not qualified for press registration and must
register as nonmembers.
Registration Refunds/Cancellations
Refunds for registration must be made in writing and postmarked by July 1, 2014. Badge and
receipt must accompany the request (except for non-U.S. xvii registrants).
Refunds cannot be made if badges are not received by the Society Office, or if postmarked after
this date. A $40 cancellation fee will be deducted from all registration payment refunds.
Attendees that cancel AFTER July 1, 2014 will not receive a refund.
Badge/Publications/Tote Bag Pickup - Grand Hall Foyer
U.S. attendees who registered prior to July 2 received their badge in advance of the meeting. All
other registrants including International registrants must go to the Symposium Registration
Counter. All attendees are required to wear their badge at all times. In addition to
being a means of identification, the name badge is required for admission to scientific sessions
and exhibits. Each registrant will receive one copy of the Program/Abstract book and one tote
bag.
Business Center
The Business Center is located on the lobby level in the Seaport Tower. It may be reached at 619232-1234 ext 4888 and is open daily, 7:00 am – 7:30 pm. Services include printing, copying,
transparencies, faxing, shipping and special services such as name tags and business cards.
Cameras/Video Recording
The use of cameras/video recording inside session rooms or among the posters is prohibited.
Mobile Devices
As a courtesy to your fellow attendees, please turn off all cell phones and beepers prior to
entering a session room.
10
Meal Functions
Meals are not included with the Symposium registration fee. Lunchtime workshops require a
$10 deposit at registration to reserve a boxed lunch.
Meeting Management Office
The Management Office is located in LaJolla A and will be open Saturday 8:00 am – 10:00 am,
Sunday – Tuesday 8:00 am – 5:00 pm, and Wednesday 8:00 am – 11:30 am.
Complimentary Wireless Internt
Symposium attendees staying at the Headquarters Hotel should have received instructions for
complimentary in-room WiFi internet access. If you were not informed or otherwise paid
for the access, please inform the hotel at checkout and they will refund the fees to you.
Complimentary WiFi access is available in the Exhibit Hall as well.
Poster Set Up & Removal
Posters can be mounted from 8:30 AM to 11:30 AM on Sunday, July 27th. Note that all
posters must be no larger than 3 feet (36 inches / 91 centimeters) to a side. Grand Hall C & D.
Posters can remain mounted until 3:30 PM on Tuesday, July 29th. Any posters
remaining after that point will be discarded.
Poster Viewing Times
Posters are on display from Sunday morning until Tuesday afternoon. During the following
shifts, exhibitors will be on hand and--during the 2 afternoon shifts--a Mix & Mingle networking
reception taking place:
SUNDAY, JULY 27TH
11:30 AM – 1:30 PM
4:30 PM – 6:30 PM
MONDAY, JULY 28TH
11:30 AM – 1:30 PM
4:30 PM – 6:30 PM
TUESDAY, JULY 29TH
11:30 AM – 1:30 PM
POSTER SESSION KEY
CELLULAR STRUCTURES - P02
CHEMICAL BIOLOGY & ENZYMOLOGY - P03
PROTEIN DEGRADATION - P04
FRONTIER HIGH- THROUGHPUT TECHNIQUES - P05
MEMBRANE PROTEINS & RECEPTORS - P06
TRANSLATION & FOLDING - P07
PROTEIN EVOLUTION - P08
PROTEINS IN ALTERED STATES - P09
PROTEINS IN DISEASE & THERAPEUTICS - P10
PROTEINS IN DYNAMIC & DRIVEN PROCESSES - P11
PROTEIN ENGINEERING & SYNTHETIC BIOLOGY - P12
Authors will be presenting posters on the following schedule:
SUNDAY 11:30 - 1:30
SUNDAY 4:30 - 6:30
Even numbered posters P02-P08
Odd numbered posters P02-P08
MONDAY 11:30 - 1:30
MONDAY 4:30 - 6:30
Even numbered posters P09-P12
Odd numbered posters P09-P12
11
TRAVEL AWARDS
The following outstanding students and early-career investigators received travel assistance
to attend the 28th Annual Symopsium of The Protein Society from The Finn Wold Travel
Awards fund and The Protein Science Young Investigator Travel Grants:*
Undergraduate Students
Angela Harper, Wake Forest University
Gabrielle Shea, Wake Forest University
Lauren Sparks, Hamline University
Mark Volker, Hamline University
Graduate Students
Keith Ballard, University of Massachusetts,
Amherst
Diego Caballero, Yale University
Alexandra Chadwick, Medical College of
Wisconsin
Genevieve Desjardins, University of British
Columbia
Maayan Eitan-Wexler, Faculty of Medicin,
Hebrew University
Keith Fraser, Rensselaer Polytechnic Institute
Gilberto Garcia, Universidad Autónoma del
Estado de Morelos
Nathan Gardner, Purdue University
Alisa Glukhova, University of Michigan
Julia Hayden, Wake Forest University
Kyle Heim, University of Florida
Michael Jacobsen, University of Utah
Agnieszka Kendrick, University of Colorado
Denver
Janelle Leuthaeuser, Wake Forest University
Carrie Marean-Reardon, Washington State
University
Tahria Najnin, University of New South Wales
Robert Newberry, University of WisconsonMadison
Olive Njuma, Auburn University
Rachel North, University of Canterbury
Satoshi Oshiro, The University Of Tokyo
Rachael Parker, Virginia Tech
Eamonn Reading, University of Oxford
Ashley Schloss, Yale University
Crystal Serano, University of Southern
Mississippi
Amber Smith, University of Michigan
Nardos Sori, Old Dominion University
Miroslava Strmiskova, University of Ottawa
Danielle Williams, Yale University
Daniel Woldring, University of Minnesota
Meng Yang, Stony Brook University
Shlomo Zarzhitsky , Ben-Gurion University of
the Negev
Huimei Zheng, State University of New York –
Upstate Medical University
Early-Career Investigators
Andisheh Abedini , New York University
Medical Center
Richard Besingi, University of Florida
Jennifer Cash, University of Michigan
Claudia Corbo, The Houston Methodist
Research Institute
Kendra Frederick, Whitehead Institute
Soon Goo Lee, Washington University in St.
Louis
Piere Rodriguez-Aliaga, University of
California Berkekey
Jennifer Seedorff, NIH/NIAID
Kaustubh Sinha, Carnegie Mellon University
Adnan Sljoka, Ryerson University/University
of Colorado Boulder
12
CHARITABLE SUPPORT
OUR MISSION
As a not-for-profit scientific and educational
organization, The Protein Society relies upon support
from the public for a vital portion of its operating budget,
and in particular to fund certain programs. The following
recent donors have the gratitude of The Protein Society
leadership and Executive Council for their generosity and
their commitment to the mission of the protein society.
The Protein Society Annual Fund
Ann E. Aulabaugh
Len Banaszak
Arpad Karsai
Edyth Malin
Abhinav Nath
Sheena Radford
Bixun Wang
The Education & Mentoring
Committees’ Funds
Jessica Bell
Penny Beuning
Norma J Greenfield
Tijana Z Grove
Blake Hill
Peter C Kahn
Jody McGinness
The Finn Wold Travel Awards Fund
Joseph Alia
Rodrigo Alejandro ArreolaBarroso
Len Banaszak
Zengyi Chang
Nidhee Chaudhary
Archana Chavan
Supratik Dutta
Anne Gershenson
Jenny P Glusker
Jacob M Goldberg
David P Goldenberg
Elizabeth Goldsmith
Mary G Hamilton
John R Helliwell
James C Hu
Yao-Te Huang
Michael N James
Joel Janin
Christos Karamitros
Andreas Matouschek
Jody McGinness
C James McKnight
Mary Jo Jo Ondrechen
Gavin Ray Owen
Arthur Palmer
Jack Preiss
George Rose
Madeline Shea
Frank Soennichsen
Melissa Starovasnik
Cynthia Stauffacher
Bernadine Wold
Marc Wold
Beulah Woodfin
*The Protein Science Young Investigator Travel Grants are sponsored by
13
DAILY PROGRAM
Day 1 - Sunday, July 27, 2014
OPEN PLENARY SESSION
8:30 AM - 9:10 AM /GRAND HALL C & D
THE 2014 HANS NEURATH AWARD TALK
8:30- 8:35
Welcome & Introduction
Protein Society President , James Bowie
8:35- 8:40
Presentation of the Hans Neurath Award*
8:40 - 9:10
2014 Neurath Award Winner Talk
The Coreography of Self-Cannibalism
James H. Hurley, University of California, Berkeley
Coffee & Refreshments
9:10 - 9:40
* SPONSORED BY THE NEURATH FOUNDATION
CONCURRENT MORNING SYMPOSIA 1
8:30 AM - 11:30 AM / LOCATION
TRANSLATION & FOLDING
CHAIR – JANE CLARKE, UNIVERSITY OF CAMBRIDGE
9:40- 9:45
9:45- 10:15
Welcome & Introduction from session Chair
Policing Secretion: How Cells Enforce Protein Quality
Control in the Endoplasmic Reticulum
Liz Miller, Columbia University
Young Investigator Talk
10:15 - 10:30
10:30 - 11:00
Continuous Tracking of Protein Folding at Microsecond
Resolution by a Line Confocal Detection of Single
Molecule Fluorescence
Satoshi Takahashi, Tohoku University
11:00 - 11:30
Intrinsically Disordered Proteins: Kinetics and Mechanism
Jane Clarke, University of Cambridge
14
DAILY PROGRAM
8:30 AM - 11:30 AM / LOCATION
BACTERIAL INTERACTIONS
CHAIR – DAVID LOW, UNIVERSITY OF CALIFORNIA- SANTA BARBARA
9:40- 9:45
9:45- 10:15
The Gram-negative Cell Envelope as Seen by Protein
Antibiotics
Colin Kleanthous, University of Oxford
10:15 - 10:30
10:30 - 11:00
Bacterial Proteins that Modulate Host Membrane
Transport Pathways
Craig Roy, Yale School of Medicine
11:00 - 11:30
Protein Interactions Regulating Self/Non-self-recognition
in Bacterial Contact-Dependent Growth Inhibition
David Low, University of California – Santa Barbara
LUNCH 11:30 - 1:30
 EXHIBIT HALL OPEN
 POSTER DISPLAYS OPEN
 EXHIBITOR WORKSHOP - GE HEALTHCARE (NOON - 1 PM, GASLAMP A):
“EW I-47 NEW AMERSHAM™ 600 IMAGER SERIES (CCD-BASED CAMERA SYSTEMS)”
MENTORING COMMITTEE WORKSHOP:
“HOW TO IMPROVE YOUR WRITING”
Organized by the Protein Society Mentoring
Committee, this lunchtime workshop will focus on
how to improve your writing skills with focus on
how to prepare an impactful abstract. Participants
will learn a framework to improve the clarity and
crispness of their communication.
NOON - 1 PM / GASLAMP C&D
UNDERGRADUATE STUDENT RESEARCH
SESSION
3 promising undergrads will be selected by The
Education Committee to present their work,
gaining visibility, experience, and making valuable
connections for graduate school and professional
development. Support future protein science
leaders and stop by!
NOON - 1 PM / BANKERS HILL
15
DAILY PROGRAM
CONCURRENT AFTERNOON SYMPOSIA1
1:30 PM – 4:30 PM / GRAND HALL C
FRONTEIR HIGH THROUGHPUT TECHNIQUES
CHAIR – ED MARCOTTE, UNIVERSITY OF TEXAS - AUSTIN
1:00 - 1:35
1:35 - 2:05
2:05 - 2:20
2:20 - 2:50
2:50 - 3:15
3:15 - 3:45
3:45 - 4:00
4:00 - 4:30
Ribosome profiling reveals surprises in genome decoding
in meiosis
Gloria Brar, University of California - Berkeley
Visualizing Transcription Pausing and Backtracking
Genome-wide at Nucleotide Resolution
L. Stirling Churchman, Harvard Medical School
Structural Analysis of Proteins in Native Environments,
Fact or Fiction?
Juri Rappsilber, University of Edinburgh
A Mass-Spectrometry-Based Map of Universally-Shared
Animal Protein Complexes
Ed Marcotte, University of Texas - Austin
CONCURRENT AFTERNOON SYMPOSIA2
1:30 PM – 4:30 PM / GRAND HALL D
PROTEINS IN ALTERED STATES
CHAIR – JULIE FORMAN-KAY, UNIVERSITY OF TORONTO
1:00 - 1:35
1:35 - 2:05
2:05 - 2:20
Protein Aggregation Done Right: The Biogenesis of
Functional Amyloids
Matthew Chapman, University of Michigan
16
DAILY PROGRAM
2:20 - 2:50
2:50 - 3:15
3:15 - 3:45
3:45 - 4:00
4:00 - 4:30
Structural Malleability of Intrinsically Disordered Proteins
Underlying Alternative Functional States
Peter Tompa, Vrije Universiteit Brussel
Amyloid Assemblies and their Interactions With Cellular
Components
Helen Saibil, Birkbeck College
Dynamic Complexes, Folding and Phase Separation of
Disordered Proteins in Biological Regulation
Julie Forman-Kay, The Hospital for Sick Children
Day 2 - Monday, July 28, 2014
8:30 AM - 11:30 AM / GRAND HALL C
PROTEIN EVOLUTION
CHAIR - JOSEPH P. NOEL, SALK INSTITUTE FOR BIOLOGICAL STUDIES / HHMI
8:30 - 8:35
8:35 - 9:05
Evolution in a Test Tube Yields De Novo Enzymes with
Unusual Structure and Dynamics
Burckhard Seelig, University of Minnesota
9:05 - 9:20
9:20 - 9:50
Sequence-function-fitness Landscapes Viewed by
Massively Parallel Sequencing Approaches
Dan Bolon, University of Massachusetts Medical School
9:50 - 10:15
17
DAILY PROGRAM
10:15 - 10:45
10:45 - 11:00
11:00 - 11:30
Evolution of Novel Components of the Bacterial Flagellar
Motor
Morgan Beeby, Imperial College London
The Remarkable Pliability and Promiscuity of Specialized
Metabolism
Joseph P. Noel, Salk Institute for Biological Studies / HHMI
8:30 AM - 11:30 AM / GRAND HALL D
PROTEINS IN DISEASE & THERAPUETICS*
CHAIR - PAMELA BJORKMAN, CALIFORNIA INSTITUTE OF TECHNOLOGY / HHMI
8:30 - 8:35
8:35 - 9:05
Structural Basis of Broad Neutralization of Viral
Pathogens
Ian Wilson, The Scripps Research Institute
9:05 - 9:20
9:20 - 9:50
9:50 - 10:15
10:15 - 10:45
10:45 - 11:00
11:00 - 11:30
The HIV-1 viral spike: Conformational Machine For Entry
and Evasion
Peter Kwong, Vaccine Research Center, Niaid/Nih
Immune Sensing of Vitamin B Metabolites
Jamie Rossjohn, Monash University
Engineering Improved Antibodies Against HIV
Pamela Bjorkman, California Institute of Technology / HHMI (Chair)
photo: Micheline Pelletier / Gamma
18
DAILY PROGRAM
LUNCH 11:30 - 1:30
 EXHIBITOR WORKSHOP - NANOTEMPER TECHNOLOGIES (NOON - 1 PM,
GASLAMP A):
“SOME LIKE IT HOT: DETERMINATION OF BIOMOLECULAR INTERACTIONS USING MICROSCALE
THERMOPHORESIS)”
“HOW TO GIVE AN EFFECTIVE TALK”
EDUCATOR’S LUNCHEON
ORGANIZED BY THE PROTEIN SOCIETY MENTORING
COMMITTEE, THIS LUNCHTIME WORKSHOP WILL
“ASSESSING STUDENT UNDERSTANDING OF
FOUNDATIONAL CONCEPTS OF PROTEIN
STRUCTURE AND FUNCTION”
FOCUS ON HOW TO IMPROVE YOUR PRESENTATION
SKILLS, AND TIPS ON HOW TO GIVE MEANINGFUL
PRESENTATIONS. GUIDELINES FOR AVOIDING
COMMON MISTAKES AND STRATEGIES FOR RELAYING
A CLEAR MESSAGE WILL ALSO BE COVERED.
CONCURRENT AFTERNOON SYMPOSIA 1
1:00 PM - 4:30 PM / GRAND HALL C
CHEMICAL BIOLOGY & ENZYMOLGY
CHAIR –BENJAMIN CRAVATT III, THE SCRIPPS RESEARCH INSTITUTE
1:00 - 1:35
1:35 - 2:05
Spatially-Resolved Proteomic Mapping of Mitochondria in
Living Cells Using an Engineered Peroxidase Reporter
Alice Ting, Massachusetts Institute of Technology
2:05 - 2:20
2014 Protein Science Best Paper Speaker
Mark Landau, Yale University
2:20 - 2:50
Structure-Based Screens for Protein De-orphanization
Brian Shoichet, University of Toronto / University of California – San
Francisco
2:50 - 3:15
3:15 - 3:45
Molecular DNA Devices in Living Systems
Yamuna Krishnan, National Center for Biological Sciences
19
DAILY PROGRAM
3:45 - 4:00
4:00 - 4:30
Activity-based Proteomics - Applications for Enzyme and
Inhibitor Discovery
Benjamin Cravatt III, The Scripps Research Institute
CONCURRENT AFTERNOON SYMPOSIA 2
1:00 PM - 4:30 PM / GRAND HALL D
CHEMICAL STRUCTURES
CHAIR –REBECCA HEALD, UNIVERSITY OF CALIFORNIA - BERKELEY
1:00 - 1:35
1:35 - 2:05
New Insights Into Microtubule Mechanics
Manuel Théry, LPCV / iRTSV / DSV / CEA
2:05 - 2:20
2:20 - 2:50
2014 Lorne Conference on Protein Structure and
Function Speaker
Kaye Truscott, Latrobe University
2:50 - 3:15
3:15 - 3:45
A Protein Interaction Network that Directs Human
Cytoplasmic Dynein to Microtubule Ends
Thomas Surrey, LRI - CRUK
3:45 - 4:00
4:00 - 4:30
Mechanisms of Mitosis and Intracellular Scaling in
Xenopus
Rebecca Heald, University of California - Berkeley
20
DAILY PROGRAM
Day 3 - Tuesday, July 29, 2014
8:30 AM - 11:30 AM /GRAND HALL C
PROTEIN ENGINEERING & SYNTHETIC BIOLOGY
CHAIR – DEK WOOLFSON, UNIVERSITY OF BRISTOL
8:30 - 8:35
8:35 - 9:05
Please consult the Program Addendum
Chris Voigt, Massachusetts Institute of Technology
9:05 - 9:20
9:20 - 9:50
Sustaining Life With Proteins Designed De Novo
Michael Hecht, Princeton University
9:50 - 10:15
10:15 - 10:45
10:45 - 11:00
11:00 - 11:30
Metal-Directed Protein Evolution
Akif Tezcan, University of California- San Diego
De Novo Protein Structures and Assemblies by Design
Dek Woolfson, University of Bristol
PROTEINS IN DYNAMIC & DRIVEN PROCESSES
CHAIR – KLAUS HAHN, UNIVERSITY OF NORTH CAROLINA – CHAPEL HILL
8:30 - 8:35
8:35 - 9:05
Deciphering Protein Dynamics During Endocytic Budding
by Time-resolved Electron Microscopy
Maria Isabel Geli Fernandez- Penaflor, Molecular Biology Institute of
Barcelona
21
DAILY PROGRAM
9:05 - 9:20
9:20 - 9:50
The Spatial Regulation of Molecular Motors
Samara Reck-Petersen, Harvard Medical School
9:50 - 10:15
10:15 - 10:45
The Bacterial Magnesium Channel Cora – Dynamic Ways
to Translocate Divalent Cations
Emil F. Pai, University of Toronto
10:45 - 11:00
11:00 - 11:30
Klaus Hahn, University of North Carolina-Chapel Hill
LUNCH 11:30 - 1:30
 EXHIBITOR WORKSHOP - GE HEALTHCARE (NOON - 1 PM, GASLAMP A):
“EW III-49 MULTIMODAL CHROMATOGRAPHY: A NEW GENERATION OF HIGH SELECTIVITY
MEDIA FOR CHALLENGING PURIFICATIONS”
WORKSHOP: “WIKIPEDIA FOR PROTEIN
SCIENTISTS”
This guided workshop, led by Wiki expert Emily
Temple-Wood, will be a fun, hands-on course on
wiki-editing, Wikipedia in general, and tips and
tricks for improving science articles using famous
protein researchers as examples. So have
someone in mind, and bring your tablet or laptop!
“HOW TO NAVIGATE YOUR CAREER”
Organized by the Protein Society Mentoring
Committee, this lunchtime workshop will consist of a
panel of individuals with different career paths to
answer your questions. The panel will cover important
aspects including connecting with hiring
managers/search committees, preparing resumes/CVs,
interview practices, and negotiations. The panel will
also be there to discuss their experiences with
traversing their career paths.
22
DAILY PROGRAM
PLENARY AWARDS SESSION
1:30 PM - 5:50 PM /GRAND HALL C & D
1:30 - 1:35
Welcome & Introduction from session James Bowie, President
1:35 - 1:40
Presentation of The Dorothy Crowfoot Hodgkin Award*
1:40 - 2:10
2014 Hodkgin Award* Winner Talk
Judith Frydman, Stanford University School of Medicine
2:10 - 2:15
Presentation of The Christian B. Anfinsen Award
2:15 - 2:45
2014 The Anfinsen Award Winner Talk
Robert Tycko, NIDDK/NIH
2:45 - 2:50
2:50 - 3:20
Presentation of The Emil T. Kaiser Award
2014 Kaiser Award Winner Talk
Carol Fierke, University of Michigan
3:20 - 3:40
3:40 - 3:45
Presentation of The Protein Science Young Investigator Award
3:45 - 4:15
2014 Young Investigator Award Winner Talk
M. Madan Babu, MRC LMB Cambridge
4:15- 4:20
Presentation of the 2013 Hans Neurath Award**
4:20-4:50
2013 Neurath Award Winner Talk
Jennifer Doudna, University of California - Berkeley
4:50-5:00
Presentation of the Protein Society Service Awards
5:00-5:15
Presentation of the Best Poster Awards***
5:20-5:50
Presentation of the Carl Brändén Award****
Stephen White, University of California - Irvine
*SPONSORED BY GENENTECH | **SPONSORED BY THE NEURATH FOUNDATION | ***THIS ACTIVITY IS SPONSORED BY AN EDUCATIONAL GRANT FROM LILLY. FOR MORE INFORMATION CONCERNING LILLY GRANT
FUNDING PLEASE VISIT WWW.LILLYGRANTOFFICE.COM | ****SPONSORED BY RIGAKU
23
DAILY PROGRAM
Day 4 - Wednesday, July 30 2014
8:30 AM - 10:35 AM / GRAND HALL C
PROTEIN DEGRADATION
CHAIR -PLEASE CONSULT THE PROGRAM ADDENDUM
8:30 - 8:35
8:35 - 9:05
9:05 - 9:20
9:20 - 9:50
10:05 - 10:35
Ubiquitin-dependent Regulation of Proliferation and
Differentiation
Michael Rape, University of California- Berkeley
Investigating the Mechanisms of the Proteasome by
Cryo-EM
Paula da Fonseca, MRC-LMB, Cambridge
Chemical Tools for the Study of Proteolytic Processes
Matthew Bogyo, Stanford University
MEMBRANE PROTEIN & RECEPTORS*
CHAIR – DOUGLAS REES, CALIFORNIA INSTITUTE OF TECHNOLOGY / HHMI
8:30 - 8:35
8:35 - 9:05
9:05 - 9:20
9:20 - 9:50
9:50 - 10:05
Mechanism-based Tuning of Cytokine Receptor Signaling
K. Christopher Garcia, The Scripps Research Institute
From Liposomes to Fliposomes: In Vitro Reconstitution of
Lipid-Dependent Dual Topology And Post-Assembly
Topological Switching of a Membrane Protein
William Dowhan, University of Texas Houston Medical School
24
DAILY PROGRAM
10:05 - 10:35
Douglas Rees, California Institute of Technology / HHMI
CLOSING PLENARY SESSION
11:00 AM - 12:30 PM /GRAND HALL C & D
AWARD WINNER TALK
11:00- 11:05
Welcome & Introduction
Protein Society President , James Bowie
11:05- 11:10
Presentation of the Stein and Moore Award
11:10 - 12:30
2014 Stein and Moore Award WINNER TALK
Nikolaus Pfanner, University of Freiburg
END OF PROGRAM
AFTER THE MEETING....
25
POSTER SESSIONS
Grand Hall A & B
Author Presentation Schedule:
Sunday, 07/27/2014
P02 - P08 Even Numbers 11:30 AM-01:30 PM
P02 - P08 Odd Numbers 4:30 PM – 6:30 PM
Cellular Structures
POST 02-224 Dissecting the repetitive C-terminal
domain of RNA polymerase II Stephen Fuchs,
Mohammad Mosaheb, Summer M. Morrill
POST 02-225 Bacterial Microcompartments:
Substrate Transport through Shell Pores
Sunny Chun, Chiranjit Chowdhury, Jiyong Park, Kendall
N. Houk, Thomas A. Bobik, Todd O. Yeates
POST 02-226 Sequence Signature for Recognition of
Histone H3 Arginine (R2) Francisca Essel, Suvobrata
Chakravarty, Tao Lin
Monday, 7/28/2014
P09-P12 Even Numbers 11:30 AM-01:30 PM
P09-P12 Odd Numbers 4:30 PM – 6:30 PM
POST 03-232 Directed evolution of a highly sensitive
peroxidase reporter and application to electron
microscopic visualization of MICU1.
Stephanie S. Lam, Jeffrey D. Martell, Kimberli J. Kamer,
Vamsi K. Mootha, Alice Y. Ting
POST 03-233 Characterization of the alphaproteobacteria Wolbachia pipientis protein
disulphide machinery reveals a regulatory mechanism
absent in gamma-proteobacteria
Patricia M. Walden, Maria A. Halili, Julia K. Archbold,
Fredrik Lindahl, David P. Fairlie, Kenji Inaba, Jennifer L.
Martin
POST 02-227 Microfluidic control and in situ
monitoring of microtubule maturation kinetics
provide evidence for a new stabilising cap model of
dynamic instability Christian Duellberg, Nicholas
Cade, David Holmes, Thomas Surrey
POST 03-234 The metal-dependent FAD
pyrophosphatase/FMN transferase activity of
periplasmic flavin-trafficking protein (Ftp): a potential
role in flavoprotein biogenesis Ranjit K. Deka, Chad A.
Brautigam, Wei Z. Liu, Diana R. Tomchick, Michael V.
Norgard
POST 02-228 Investigating the function of Suppressor
of IKK-epsilon Sean W. McKinley, Kenneth F. Lawrence,
Jessica K. Bell
POST 03-235 Characterization of hyperthermophilic
TyrA enzymes involved in aromatic amino acid
biosynthesis Irina Shlaifer, Joanne L. Turnbull
POST 02-229 Structure of BDBT reveals a role for
noncanonical FK506 binding protein in regulation of
the fly circadian clock. Boadi Agyekum, Jin-Yuan Fan,
Anandakrishnan Venkatesan, Jeffrey Price, Samuel
Bouyain
POST 03-236 Probing novel antibiotic targets within
sialic acid catabolism Rachel A. North, Sarah A.
Kessans, Hironori Suzuki, Michael D. Griffin, Renwick C.
Dobson
POST 02-230 The PKD-related proteins ANKS6, BICC1,
and ANKS3 form a SAM domain interaction network
Catherine N. Leettola, Mary J. Knight, Duilio Cascio,
James U. Bowie
Chemical Biology & Enzymology
POST 03-231 The variations of protein splicing:
regulation and mechanism of non-canonical inteins
Kenneth Mills, Julie N. Reitter, Michael Nicastri, Jennie
Williams, Kathryn Colelli, Michelle Marieni
POST 03-237 The Role of Protein Glycosylation in
Laccases from Lentinus sp. Wei-Chun Liu, Manuel
Maestre-Reyna, Wen-Yih Jeng, Cheng-Chung Lee, ChihAn Hsu, Tuan-Nan Wen, Andrew H.-J. Wang, Lie-Fen
Shyur
POST 03-238 FBP17 plays a role in the morphological
control by regulating the activity of Rho subfamily
GTPase CDC42 Jun Zhang, Lin Ming-ming, Zhang Qianying, Wang Yun-hong, Li Xin
POST 03-239 Semi-Synthesis and Applications of
Fluorophore/Thioamide pairs Containing Proteins
Solongo Batjargal, E. James Petersson
POST 03-240 Computational Design of an Unnatural
Amino Acid Dependent Metalloprotein with Atomic
26
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
Level Accuracy Jeremy Mills, Sagar D. Khare, Jill M.
Bolduc, Farhad Forouhar, Vikram K. Mulligan, Scott
Lew, Jayaraman Seetharaman, Liang Tong, Barry L.
Stoddard, David Baker
POST 03-241 Acrolein-modified High Density
Lipoproteins Promote Atherogenesis Alexandra
Chadwick, Rebecca L. Holme, Yiliang Chen, Kirkwood A.
Pritchard, Daisy Sahoo
POST 03-242 Control of protein production and virus
replication by pharmacological blockade of degron
detachment Hokyung K. Chung, Conor Jacobs, Yunwen
Huo, Jin Yang, Stefanie A. Krumm, Richard K. Plemper,
Rodger Y. Tsien, Michael Z. Lin
POST 03-243 Understanding the metabolism of
enteric pathogen Campylobacter jejuni Adnan Ayna,
Peter Moody
POST 03-244 Chitosan-binding modules (CBM32) of a
chitosanse from Paenbacillus sp. IK-5 --- Amino acid
residues responsible for chitosan binding--Shoko Shinya, Takayuki Ohnuma, Hisashi Kimoto, Hideo
Kusaoke, Tamo Fukamizo
POST 03-245 Crystal structure of a family GH18 (class
V) chitinase from cycad, Cycas revolta ---- structural
factors controling the transglycosylation activity of
the enzyme Tamo Fukamizo, Naoyuki Umemoto,
Takayuki Ohnuma, Toki Taira, Tomoyuki Numata
POST 03-246 Split intein mediated peptide cyclization
Shubhendu Palei, Henning D. Mootz
POST 03-247 Exploring the Morpheein Forms of B.
cenocepacia HMG-CoA Reductase Riley Peacock,
Michelle Brajcich, Courtney Boyd, Jeffrey Watson
POST 03-248 Dynamic Functional Switch in Poliovirus
3C Protease Yan M. Chan, David D. Boehr
POST 03-249 Lighting the Cellular Fuel Gauge with
Fluorescent Sensors for Imaging Single-Cell
Metabolism Mathew Tantama, Juan Ramón MartínezFrançois, Rebecca Mongeon, Gary Yellen
Monday, 7/28/2014
POST 03-250 Structural analysis and molecular
dynamics of the self-sufficient P450 CYP102A5 and
CYP102A1: A combined computational/experimental
approach to increase the efficiency of biocatalyst
engineering. Maximilian Ebert, Brahm Yachnin,
Guillaume Lamoureux, Albert Berghuis, Joelle Pelletier
POST 03-251 Isolation and characterization of proline
specific dipeptidyl peptidase IV from the Tenebrio
molitor larval midgut Valeriya F. Sharikova, Irina
Goptar, Yulia Smirnova, Brenda Oppert, Irina Filippova,
Elena Elpidina
POST 03-252 Characterization of hydrolytic enzymeproducing bacteria isolated from paper mill
Manel Ghribi, Fatma Meddeb-Mouelhi, Marc
Beauregard
POST 03-253 Isolation of NRPS and PKS Gene Clusters
from Soil Microbes Danielle N. O'Hara, Connor P. Craig
POST 03-254 Treatment of kraft pulp with enzymes
for improving beatability and physical properties
Li Cui, Fatma Meddeb, Marc Beauregard
POST 03-255 Structural and biochemical investigation
of the intramolecular interactions of ceramide
transfer protein Jennifer Prashek, Seungkyung Kim,
Xiaolan Yao
POST 03-256 Evaluating interpretation of B-factors for
collective motion modeling Edvin Fuglebakk, Nathalie
Reuter, Konrad Hinsen
POST 03-257 Structure/Function Relationships in
Carboxylesterase EstGtA2 from Geobacillus
thermodenitrificans Jessica K. Moisan, Fatma
Meddeb-Mouelhi, Marc Beauregard
POST 03-258 Up-regulation of Rich1 causes S-phase
arrest and reduces cell adhesion in epithelial cells
Lin Ming-ming, Zhang Qian-ying, Wang Yun-hong, Li
Xin, Zhang Jun
POST 03-259 Tracking wood fibers decrystallization
with carbohydrate binding module Yannick Hébert-
27
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
Monday, 7/28/2014
Ouellet, Vinay Khatri, Fatma Meddeb-Mouelhi, Marc
Beauregard
organisms Jorge D. Garcia, David G. Mendoza-Cozatl,
Rafael Moreno-Sánchez
POST 03-260 Evolution of Structure and Mechanistic
Divergence in Di-Domain Methyltransferases from
Nematode Phosphocholine Biosynthesis Soon Goo
Lee, Joseph M. Jez
POST 03-270 Nonlocal effects of metal ion binding at
the catalytic site of a protein-DNA complex. Kaustubh
Sinha, Michael R. Kurpiewski, Sahil Sangani, Andrew D.
Kehr, Gordon S. Rule, Linda Jen-Jacobson
POST 03-261 ERK1 and 2 - Exploring Isoform
Differences Jen Liddle, Natalie Ahn
POST 03-271 Recognition and Conversion of
Flunitrazepam by Cyp3A4 is Altered by Caffeine
Mark Volker, Lauren Sparks, Larry R. Masterson
POST 03-262 Chemical Modification of MitoNEET
Megan Laffoon, Michael Menze, Mary Konkle
POST 03-263 Biochemical basis for the extended
spectrum Cephalosporinase activity of a clinical AMPC
β-Lactamase variant Jozlyn R. Clasman, Brianna M.
Jackman, Cynthia M. June, Rachel A. Powers, David A.
Leonard
POST 03-264 Common substitutions enhance the
Carbapenemase activity of OXA-51-Like Class D βLactamases from Acinetobacter SPP. Joshua M.
Mitchell, David A. Leonard
POST 03-265 Exploring the Potential of Arylboronic
Acids as Inhibitors of OXA-24 β-lactamase Josephine P.
Werner, Rachel A. Powers
POST 03-266 Structure-based Discovery of a Novel
Inibitor of OXA-1 β-lactamase Leslie A. Wyman, Neil V.
Klinger, Rachel A. Powers
POST 03-272 Fluorogenic Probes for Mycobacterial
Esterase Profiling Katie Tallman, Kimberly Beatty
POST 03-273 Visualizing cell interactions with
genetically encoded bioluminescent tools
Krysten A. Jones, David Li, Elliot E. Hui, Mark A.
Sellmyer, Jennifer A. Prescher
POST 03-274 Specific Inhibition of Enolase From
Entamoeba histolytica Normande Carrillo-Ibarra, Cesar
Augusto Sandino Reyes-Lopez, Jose Correa-Basurto,
Elibeth Mirasol Melendez, Claudia Guadalupe Benitez
Cardoza
POST 03-275 Effects of Intrauterine and Extrauterine
Exposure to 1800 MHz GSM-Like Radiofrequency
Radiation on Liver Regulatory Enzymes Activities in
one-month-old male New Zealand Rabbits Nuray N.
Ulusu
POST 03-267 Kinetic and Biochemical Investigations
of Thermostable Acid Phosphatase from Zea mays
and Glycine max cotyledons: A therapeutically
important enzyme Nidhee Chaudhary, Subhash
Chand, Nameet Kaur
POST 03-276 Imaging Protein-Protein Interactions,
Post-Translational Modifications, and Non-Protein
Biomolecules by Correlative Light and Electron
Microscopy John T. Ngo, Daniela Boassa, Stephen R.
Adams, Thomas J. Deerinck, Sakina F. Palida, Varda
Lev-Ram, Mark H. Ellisman, Roger Y. Tsien
POST 03-268 Investigation of the dynamic amino acid
networks in a (β/α)8 barrel enzyme Jennifer M. Axe,
Kathleen F. O'Rourke, Eric M. Yezdimer, Nicole E.
Kerstetter, Xianrui Yuan, David D. Boehr
POST 03-277 Evaluating the role of peroxidatic
reducing substrates in an unusual catalase activity of
catalase-peroxidases Olive J. Njuma, Elizabeth
Ndontsa, Douglas Goodwin
POST 03-269 An uncommon phytochelatin synthase
gives hints on how to improve their catalytic
efficiency on heavy metal hyperaccumulator
POST 03-278 Pyrrolysine-Inspired Protein Cyclization
Marianne M. Lee, Tomasz Fekner, Jia Lu, Michael K.
Chan
28
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 03-279 Functional sectors involved in thermal
stability and activity in Beta-Glucosidases Fabio K.
Tamaki, Larissa C. Textor, Igor Polikarpov, Sandro R.
Marana
POST 03-280 A chemoproteomic platform to
quantitatively map targets of lipid-derived
electrophiles Chu Wang, Benjamin F. Cravatt
Monday, 7/28/2014
POST 03-288 Investigating Molecular Determinants
that Modulate the Preference of I kBs for Specific NF
kBs James D. Marion, Elizabeth A. Komives
POST 03-289 The examination of the MALDI-TOF-MS
analysis of the proteins and the peptides by use of the
sinapic acid derivatives as the new matrix.
Narumi Hirosawa, Takeshi Sakamoto, Yasushi Uemura,
Yasushi Sakamoto
POST 03-281 Protein stabilization and prevention of
protein aggregation by fungal sucrase of
Termitomyces clypeatus and application in
biotechnology and biomedical research
Suman Khowala, Sudeshna Chowdhury, Sanjeeta
Tamang, Sangita Majumdar, Rajib Majumder
POST 03-290 Sequential Phosphorylation of SIKE by
TBK1 Hyejin Park, SoHo Kim, Jessica Bell, Ellis Bell
POST 03-282 Using Multiwavelength Collisional
Quenching to Investigate the Effects of Arginine and
Inhibitors on inducible Nitric Oxide Synthase
Rachel Jones, Ellis Bell
POST 03-292 Multiwavelength Collisional Quenching
to Study Ligand Protein Interactions in Glutamate
Dehydrogenase Chun Li, Ellis Bell
POST 03-283 Enzyme Active Sites May Extend Further
than We Thought Lisa Ngu, Penny J. Beuning, Mary Jo
Ondrechen
POST 03-284 Nucleotide
pyrophosphatase/phosphonuclease possesses the
zeatin cis-trans isomerase activity in vitro
Tomáš Hluska, Michaela Baková, René Lenobel, Marek
Šebela, Petr Galuszka
POST 03-285 Exploring the Trigger for Cooperativity at
the Subunit Interface of Malate Dehydrogenase
Jacqunae Mays, Ellis Bell
POST 03-286 Redox control of Protein Arginine
Methyltransferase 1 (PRMT1) activity Yalemi Morales,
Damon Nitzel, Owen Price, Shanying Gui, Joan Hevel
POST 03-287 Structural and biochemical
characterization of Thielavia terestris Cutinase (TtC)
Abhijit N. Shirke, Danielle A. Basore, Evan Baugh, An
Su, Glen Butterfoss, George I. Makhatadze, Christopher
Bystroff, Richard A. Gross
POST 03-291 Effects of Phosphorylation on the
Structure & Stability of SIKE Clara Kerckhove, Jessica
Bell, Ellis Bell
POST 03-293 Mapping the binding sites of class D
beta-lactamases for inhibitor design and discovery
Joshua M. Mitchell, Rachel Powers
POST 03-294 Determining the Effects of MitoNEET on
Cellular Dehydrogenase Activity Sarah Banister,
Matthew Woodruff, Paige Birge, Michael Menze, Mary
Konkle
POST 03-295 In silico and kinetic studies to verify the
potency of α-glucosidase inhibitors isolated from
Morus alba L. Shakeel Ahmad, Akash Chaudhary,
Shadab Ahmad, Mohd. Tashfeen Ashraf
POST 03-296 Molecular Dynamic Studies of the
Reductase Domain of Polyketide Synthase from the
Myxobacterium Stigmatella Aurantiaca
Andrew J. Schaub, Jesus Barajas, Ray Luo, Shiou-Chaun
(Sheryl) Tsai
POST 03-297 Mechanisms regulating ribosome
biogenesis by AKT and c-MYC
Simone Woods, Colin House, Gretchen Poortinga, Ross
Hannan, Katherine Hannan
29
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 03-298 Highly selective peptide substrates for
the assay of cysteine peptidases from the C1 family
Elena N. Elpidina, Irina Y. Filippova, Tatiana A.
Semashko, Elena A. Vorotnikova, Valeriya F. Sharikova,
Konstantin S. Vinokurov, Lyndsey Fallis, Brenda Oppert
POST 03-299 Collagen Determination and Its
Application for the Authentication of Tortoise Shell
Linqiu Li, Hon-Yeung Cheung
POST 03-300 The B-domain-half-deleted recombinant
Coagulation factor VIII (FVIII) shows much higher
coagulation activity compared with B-domain-deleted
recombinant ones Zhang Jun, Zhu Chong-yang, Wen
Quan, Lin Ming-ming
POST 03-301 Novel Heterotetrameric Enzymes in
Cholesterol Metabolism from Mycobacterium
tuberculosis Meng Yang, Kip Guja, Miguel Garcia-Diaz,
Suzanne Thomas, Nicole Sampson
*
POST 03-302 Energetic contribution of n→π
interactions to PII conformations of model peptides
Liu He, Zhengshuang Shi
POST 03-303 The secretion and expression of a series
of B domain truncated recombinant coagulation
factor VIII in hepatocyte Zhang Jun, Zhu Chong-yang,
Zhang Qian-ying, Lin Ming-ming, Wen Quan, Li Xin
POST 03-304 Crystal structure studies of dipeptidyl
aminopeptidase BII from Pseudoxanthomonas
mexicana WO24. Yasumitsu Sakamoto, Yoshiyuki
Suzuki, Ippei Iizuka, Chika Tateoka, Saori Roppongi,
Mayu Fujimoto, Koji Inaka, Hiroaki Tanaka, Mika
Masaki, Kazunori Ohta, HIrofumi Okada, Takamasa
Nonaka, Yasushi Morikawa, Kazuo T. Nakamura,
Wataru Ogasawara, Nobutada Tanaka
POST 03-305 Protein Recognition by Multivalent
Fluorescent Molecular Sensors David Margulies
POST 03-306 Assessing Student Understanding of
Foundational Concepts of Protein Structure and
Function Ellis Bell
Monday, 7/28/2014
POST 03-307 Dietary condensed tannins and their
effect on microbial protein and theoretical methane
yield in ruminal fermentation on cattle in the
northern Mexico Ericka Fabiola Luisillo-Quiñones,
Gerardo A. Pámanes-Carrasco, Christian A. HernándezVázquez, Zaira J. Romo-Astorga, Esperanza Herrera
POST 03-308 Regulation and Inter-Domain
Communications in C-terminal Src Kinase (CSK)
Sulyman Barkho, Levi C. Pierce, Joseph A. Adams,
Patricia A. Jennings
POST 03-309 Site-Specific Molecular Recognition of
Proteins by Synthetic Receptors Adam Urbach
POST 03-310 Structure of Dihydromethanopterin
Reductase: Redox Transfer in a Cubic Protein Cage
1
Dan E. McNamar , Duilio Cascio, Julien Jorda, Cheene
Bustos, Tzu-Chi Wang, Madeline E. Rasch, Thomas A.
Bobik, Todd O. Yeates
POST 03-311 The Linkage Between Folding
Cooperativity and Allostery: Glutamate
Dehydrogenase Angela Tata, Grace Kingdom, Ellis Bell
POST 03-312 Hydrogen exchange of disordered
proteins in living cells Austin E. Smith, Zijian Zhou,
Gary J. Pielak
POST 03-313 Assessment of UCH-L3 Substrate
Selectivity using Engineered Ubiquitin Fusions with
Variable Linker Lengths Peter Suon, John J. Love
POST 03-314 Identification, Characterization, and
Modification of Fatty Acid Alkyl Esterases Found in
Staphylococcus aureus Benjamin D. Saylor, John J.
Love
Protein Degradation
POST 04-315 Apoptotic protein Bax is regulated by the
ubiquitin-proteasome pathway Kwang-Hyun Baek, SoRa Kim, Jin-Ok Kim
POST 04-316 Discovery and characterization of small
molecule fragments that bind and inhibit the
30
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
Ubiquitin Specific Protease 7 (USP7) Paola Di Lello,
Terry Crawford, Kurt Deshayes, Joy Drobnick, Jake
Drummond, James Ernst, Lorna Kategaya, Michael
Kwok, Cuong Ly, Till Maurer, Jeremy Murray, Chudi
Ndubaku, Rich Pastor, Lionel Rouge, Structural Biology
Expression group, Vickie Tsui, Ray Zhao, Kerry Zobel,
Ingrid Wertz
POST 04-317 Polyglycine Hydrolases Secreted by
Pathogenic Fungi Todd A. Naumann, Donald T.
Wicklow, Todd J. Ward, Michael J. Naldrett, Neil P.
Price
POST 04-318 Higher-order assembly architecture of
the AAA+ protease Lon reveals a new regulatory
mechanism for substrate specificity Tejas Kalastavadi,
Ellen Vieux, Breann Brown, Tania Baker
POST 04-319 A robust assay for protein unfolding by
AAA+ molecular machines Vladimir Baytshtok, Tania A.
Baker, Robert T. Sauer
POST 04-320 Mitochondrial Lace1 ATPase
Lukas Stiburek, Jana Cesnekova, Josef Houstek, Jiri
Zeman
POST 04-321 The structure of the human hybrid
proteasome Edward Morris, Fabienne Beuron, Paula
da Fonseca
POST 04-322 Are the Precursor Sequences of Thiol
Proteases Related to Thiol Protease Inhibitors
Meron Tarekegn, David Harry, Kelsey Kines, Ellis Bell
POST 04-323 Thiol Proteases & Thiol Protease
Inhibitors in C Elegans Kelsey Kines, David Harry,
Meron Tarekegn, Ellis Bell
POST 04-324 Structural Analysis of Poly-SUMO Chain
Recognition by RNF4-SIMs Domain
Chia-Hsiuan C. Kung, Mandar T. Naik, Hsiu-Ming Shih,
Tai-huang Huang
POST 04-325 Misfolded Proinsulin Retrotranslocation
for Proteasome-dependent Degradation in the
Cytosol Can Be Modulated by Altering the
Monday, 7/28/2014
Endoplasmic Reticulum Lumenal Composition PenJen Lin
Frontier High-Throughput Techniques
POST 05-326 Virotrap: Abducting Protein Complexes
from Mammalian Cells Sven Eyckerman, Kevin Titeca,
Emmy Vanquickelberghe, Annick Verhee, Noortje
Samyn, Delphine De Sutter, Evy Timmerman, Kris
Gevaert, Jan Tavernier
POST 05-327 Global measurement of protein
localization in C. elegans with tissue and subcellular
specificity Aaron Reinke, Eric Bennett, Emily Troemel
POST 05-328 Broad Scope and Coverage of
Functionally Relevant Groups for Sequences in the
Enolase Superfamily Brian Westwood, Stacy Knutson,
Janelle Leuthaeuser, Patricia Babbitt, Jacquelyn Fetrow
POST 05-329 A molecular toolkit for single molecule
peptide sequencing Jim Havranek, Ben Borgo
POST 05-330 Novel Solubility Fusion Partners High
Throughput System to Produce Soluble Proteins
Saurabh Sen, Eric Steinmetz, Sally Floyd, David Mead,
Mark Maffitt
POST 05-331 Elucidating the global metabolic
regulatory role of prokaryotic enzyme posttranslational modifications through systems biology
analysis Nathan E. Lewis, Roger Chang, Chen Yang,
Hooman Hefzi, Bernhard Palsson, George Church
Membrane Proteins & Receptors
POST 06-332 What’s on the Menu: Identification of
the Hydrocarbon Transport Systems as a first step in
Marine Oil-Degradation by Alcanivorax borkumensis
Swapnav Deka, Chad Brautigam
POST 06-333 Keeping in touch: T-cadherin impedes
dissociation of adiponectin receptor 1 dimers
31
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
Tobias Leimer, David Kosel, Karin Mörl, Barbara
Ranscht, Annette G. Beck-Sickinger
POST 06-334 A new rigidity-based model for allosteric
communication in G-protein coupled receptors.
Adnan Sljoka
POST 06-335 Keeping it simple: The construction of
biologically active proteins with minimal chemical
diversity Erin N. Heim, Jez L. Marston, Kelly M. Chacon,
Lisa M. Petti, Daniel DiMaio
Monday, 7/28/2014
POST 06-343 Development of a quantitative, realtime, label-free assay for ligands interacting with the
HIV Envelope glycoprotein in virus-like particles
Jennifer Seedorff, Edward Berger
POST 06-344 Silicon transport in diatoms: the key to
unlocking their full potential? Sarah Ratcliffe, Michael
Knight, Laura Senior, Paul Curnow
POST 06-336 Characterization of the second
immunoglobulin domain (Ig2) of the human receptor
CRTAM/CD355 and its role in the formation of dimer
Juan C. Barragan-Galvez, Luis Brieba-Castro, Vianney
Ortiz-Navarrete
POST 06-345 Mechanistic studies of the Alternative
Complex III from the photosynthetic bacterium
Chloroflexus aurantiacus Erica L. Majumder, Robert E.
Blankenship
POST 06-346 Structure and function of the diatom
silicon transporter Michael Knight, Laura Senior, Sarah
Ratcliffe, Paul Curnow
POST 06-337 Expression, purification and functional
refolding of human olfactory receptor expressed in
Escherichia coli Heehong Yang, Sae Ryun Ahn, Tai
Hyun Park
POST 06-347 Intermolecular interactions between the
intracellular domains of Arabidopsis CRINKLY4 (ACR4)
receptor-like kinase and homologs. Matthew R.
Meyer, Shweta Shah, Gururaj A. Rao
POST 06-338 The importance of CD4 allostericity for
interaction with HIV glycoprotein 120 Nichole M.
Cerutti, Vinesh Jugnarain, Alexio Capovilla
POST 06-348 Expression, purification and
reconstitution of the aromatic acid transporter PcaK
Christian Pernstich, Laura Senior, Katherine A.
MacInnes, Marc Forsaith, Paul Curnow
POST 06-339 Structural characterization of the major
pilin subunit from the bacterial nanowires of
Geobacter sulfurreducens Patrick N. Reardon, Karl T.
Mueller
POST 06-340 X-ray structures reveal bent
conformation for all CNTN family members
Roman M. Nikolaienko, Samuel E. Bouyain
POST 06-349 Insight into receptor-active
conformation of apolipoprotein E revealed by XL-MS:
new clues for a putative importance of helix 4
elongation in receptor recognition. Nicolas Henry,
Stéphanie Deroo, Florian Stengel, Eva-Maria Krammer,
Rouslan Efremov, Guy Vandebussche, Martine Prevost,
Ruedi Aebersold, Vincent Raussens
POST 06-341 Visualization of HIV-1 envelope
glycoprotein in live cells by labeling it with GFP in the
gp120 subunit Shuhei Nakane, Aikichi Iwamoto, Zene
Matsuda
POST 06-350 The Little Lipid That Could: Elucidating
the Effects of Small Amounts of Phosphatidic Acid on
the α-Synuclein Membrane Interaction
Sara K. Hess, Jennifer C. Lee
POST 06-342 Hemifusion induced by the HA2 subunit
of influenza virus hemagglutinin: Respective major,
moderate, and minor contributions of the soluble
ectodomain, fusion peptide, and transmembrane
regions.
Punsisi Upeka Ratnayake
POST 06-351 Structure-based Analysis of Protein
Modifications in G Protein Signaling - A New
Approach to Prioritize PTMs in a Protein Complex
Henry Dewhurst, Shilpa Choudhury, Matthew P. Torres
32
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 06-352 Molecular characterization of the
Arabidopsis CRINKLY4 receptor-like kinase (ACR4)
intracellular domain coupled with transmembrane
domain Shweta Shah, Matthew R. Meyer, Gururaj Rao
POST 06-353 Improved fusion protein strategies for
crystallization of G-protein coupled receptors
Yi Zheng, Ling Qin, Lauren Holden, Chunxia Zhao, Tracy
Handel
POST 06-354 Biochemical characterization of
interaction between the Arabidopsis receptor-like
kinase (ACR4) and phosphatase, PP2A3. Priyanka
Sandal, Matthew R. Meyer, Petra E. Gilmore, Ried
Townsend, Aragula G. Rao
POST 06-355 The proteomic signature of the
LeukoLike Vector unveils the presence of molecules
able to improve self-tolerance of the drug delivery
systems Claudia Corbo, Alessandro Parodi, Roberto
Molinaro, Michael Evangelopoulos, David A. Engler,
Shilpa Scaria, Francesco Salvatore, Anthony C. Engler,
Ennio Tasciotti
POST 06-356 Crystal structure of phosphate-bound
V1-ATPase of Enterococcus hirae Kano Suzuki, Kenji
Mizutani, Yoshiko Ishizuka-Katsura, Takaho Terada,
Mikako Shirouzu, Shigeyuki Yokoyama, Ichiro Yamato,
Takeshi Murata
POST 06-357 Structural analysis for the interaction of
sialic T antigen glycopeptide of HSV-1 with entry
receptor PILRα Takao Nomura, Jiro Sakamoto, Fumina
Oosaka, Kosuke Kakita, Atsushi Furukawa, Masahiro
Anada, Shunichi Hashimoto, Kimiko Kuroki, Toyoyuki
Ose, Hisashi Arase, Takashi Saitoh, Katsumi Maenaka
POST 06-358 Structural snapshots of the α-helical
pore-forming toxin FraC reveal the molecular basis of
its activation in membranes. Koji Tanaka, Jose M.
Caaveiro, Kouhei Tsumoto
POST 06-359 Membrane proteins bind lipids
selectively to modulate their structure and function
Eamonn Reading, Art Laganowsky, Timothy M. Allison,
Carol V. Robinson
Monday, 7/28/2014
POST 06-360 NMR Studies of G-Protein Coupled
Receptors Jasmina Radoicic, Sang Ho Park, Anna De
Angelis, Bibhuti Das, Sabrina Berkamp, Stanley J. Opella
POST 06-361 Structure Determination of Vpu from
HIV-1 by NMR Hua Zhang, Eugene Lin, Stanley Opella
POST 06-362 NMR studies of a GPCR with ligand
bound: CXCR1 and Interleukin-8 Sabrina Berkamp,
Anna De Angelis, Bibhuti Das, Sang Ho Park, Mitchell J.
Zhao, Jasmina Radoicic, Stanley J. Opella
POST 06-363 Membrane Protein Folding Stability and
Kinetics in Bilayers Yu-Chu Chang, James U. Bowie
POST 06-364 Effects of Mercury Ion on the Structure
and Function of E. coli Aquaporin Z
Qingsong Lin, Hu Zhou, Lili Wang
POST 06-365 Characterization of the calcium and
membrane binding properties of the hearing related
protein otoferlin Colin P. Johnson, Murugesh
Padmanarayana, Nicole Hams, Ryan Mehl
POST 06-366 TOWARDS A CRYSTAL STRUCTURE OF
THE HIV-1 MEMBRANE PROTEIN, Vpu Arpan Deb,
William Johnson, Dustin Srinivas, Liqing Chen, Petra
Fromme, Tsafrir Leket-Mor
POST 06-367 Conformational Landscape Governing
the Constitutive Activity of GPCRs Ravinder Abrol,
Caitlin E. Scott, William A. Goddard III, Kwang H. Ahn,
Debra E. Kendall
POST 06-368 Cell-free translation systems for
biophysical and biochemical characterization of
proteins and protein complexes Feliza A. Bourguet,
Craig D. Blanchette, Nicholas O. Fischer, Paul J. Jackson,
Masood Z. Hadi, Wei He, Brian K. Kay, Kit S. Lam, Ted A.
Laurence, Zachary Rogers, John C. Voss, Matthew A.
Coleman
POST 06-369 Membrane proteins can have high
kinetic stability Robert Jefferson, Tracy Blois, James
Bowie
POST 06-370 Advancing membrane protein
crystallography using the LCLS Mark Hunter, Brent W.
33
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
Segelke, Nadia Zatsepin, Matt Coleman, W. Henry
Benner, Stefan Hau-Riege, Ching-Ju Tsai, Xiao-dan Li,
Bill Pedrini, Gebhard Schertler, Matthias Frank
Translation & Folding
POST 07-371 Elucidating the structure and dynamics
of small heat shock protein complexes using a hybrid
approach . Michelle Heirbaut, Steven Beelen, Esther
Martin, Frederik Lermyte, Tim Verschueren, Frank
Sobott, Sergei Strelkov, Stephen Weeks
POST 07-372 Achieving folding cooperativity in a
physiological environment Nathan Gardner, Chiwook
Park
POST 07-373 n→π* Interactions in Protein Structure
and Folding Robert W. Newberry, Ronald T. Raines
POST 07-374 Structural and dynamic insights about
unfolding intermediates in four amyloidogenic
immunoglobulin light chains. Gilberto Valdes-Garcia,
Cesar Millan-Pacheco, Nina Pastor
POST 07-375 Mechanistic insights into the folding of
the trefoil-knotted proteins Nicole C. Lim, Sophie E.
Jackson
POST 07-376 Intermolecular Interactions in a Blood
Clotting Mechanism Led to Protein Folding Theory
Harold A. Scheraga
POST 07-377 Molecular mechanism of nuclear
transport mediated by flexible amphiphilic proteins
Shigehiro Yoshimura, Msahiro Kumeta, Kunio Takeyasu
POST 07-378 Native state dynamics of the prion
protein probed by Hydrogen exchange and mass
spectrometry Roumita Moulick, Jayant B. Udgaonkar
POST 07-379 Mutations in the bacterial ribosomal
protein S12 influence aminoglycoside antibiotic and
ribosome dynamics Joanna Panecka, Cameron Mura,
Joanna Trylska
Monday, 7/28/2014
POST 07-380 Curious characteristics of a mutant
chaperonin GroEL with multiple cysteines in the
central cavity Tomohiro Mizobata, Shuhei Fusa,
Masashi Ikeda, Kunihiro Hongo, Yasushi Kawata
POST 07-381 Untangling ribosome biogenesis using
quantitative mass spectrometry, electron microscopy
and chemical probing Joseph H. Davis, Nikhil Jain,
Admad Jomaa, Joaquin Ortega, Robert Britton, James
R. Williamson
POST 07-382 Study of E.coli GroEL using stoppedflow analysis and circular permutation Toshifumi
Mizuta, Tatsuya Uemura, Kunihiro Hongo, Yasushi
Kawata, Tomohiro Mizobata
POST 07-383 A Global machine learning-based
scoring function for protein structure prediction
Andrzej Kloczkowski, Eshel Faraggi
POST 07-384 Effect of Mg ‘2+ in the structure and
thermal stability of enolase from Trichomonas
vaginalis Elibeth Mirasol Meléndez, Jorge L. Rosas
Trigueros, Luis G. Brieba de Castro, Rossana Arroyo
Verástegui, Claudia G. Benítez Cardoza
POST 07-385 Controlling nanostructures of insulin
amyloid fibrils using metal ions Misaki Yokoyama,
Yoshito Furuie, Motonari Tsubaki, Hiroshi Hori,
Takamasa Nishida, Kazuo Eda, Eri Chatani
POST 07-386 Structural and Thermodynamic
Characterization of the X−Dimer of Human PCadherin: Implications for Homophilic Cell Adhesion
Shota Kudo, Jose Caaveiro, Satoru Nagatoishi, Takao
Hamakubo, Tatsuhiko Kodama, Tadashi Matsuura,
Yukio Sudou, Kouhei Tsumoto
POST 07-387 A Novel Protein Fold within the Nterminus of a Streptococcal Adhesin Mediates Proper
Folding, Function, and Stability Kyle P. Heim, Paula
Crowley, Shweta Kailasan, Robert McKenna, Jeannine
Brady
POST 07-388 Folding of Collagen Heterotrimeric
Helices via Cation- π Interactions Jia-Cherng Horng,
Chu-Harn Chiang, Tang-Chun Kao
34
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 07-389 Loss of Conformational Entropy in
Protein Folding Calculated using Realistic Ensembles
(with implications to NMR-based calculations) Tobin
R. Sosnick, Michael C. Baxa, Esmael Haddadian, Karl F.
Freed
POST 07-390 Rare example of a protein where an
isolated domain is more stable than the full-length
Swati Bandi, Surinder Singh, Krishna Mallela
POST 07-391 Analysis of several monomeric mutants
of Triosephosphate Isomerase.
MIsrain E. Gurrola Acosta, Maria E. Chánez, Edgar
Vazquez Contreras
POST 07-392 Probing the Denatured State of a
Knotted Protein David J. Burban, Dominique Capraro,
Joanna Sulkowska, Patricia Jennings
POST 07-393 Transitions between different side-chain
conformations in hydrophobic residues.
Diego Caballero, Corey S. O'Hern, Lynne Regan
POST 07-394 N-terminal domain of Luciferase controls
misfolding avoidance Zackary N. Scholl, Weitao Yang,
Piotr Marszalek
POST 07-395 Direct measurement of the multimer
stabilization in the mechanical unfolding pathway of
Streptavidin Zackary N. Scholl, Piotr Marszalek
POST 07-396 ATPase domain of DnaK, Escherichia
coli Hsp70 molecular chaperone, experiences pHdependent ATPase activity upon linker binding due to
Asp194 and Glu171 Rahmi Imamoglu, Umut Gunsel,
Bulent Balta, Gizem Dinler-Doganay
Protein Evolution
POST 08-397 Structural Evidence for Antigen
Receptor Evolution Romain Rouet, David Langley,
Daniela Stock, Daniel Christ
POST 08-398 Computational prediction and
experimental characterization of a “size switch type
Monday, 7/28/2014
repacking” during the evolution of dengue envelope
protein domain III (ED3) Montasir Elahi, Keiichi
Noguchi, Masafumi Yohda, Hiroyuki Toh, Yutaka
Kuroda
POST 08-399 Evolutionary Exploitation of
Promiscuous NSAR/OSBS Enzymes Andrew McMillan,
Denis Odokonyero, Mariana Lopez, DaNae Woodard,
Ashley Brizendine, Margaret E. Glasner
POST 08-400 Active site profile-based clustering of
enolase structures and sequences Janelle Leuthaeuser,
Stacy Knutson, Brian Westwood, Patricia Babbitt,
Jacquelyn Fetrow
POST 08-401 Active site profile-based clustering of the
peroxiredoxin superfamily Angela Harper, Janelle
Leuthaeuser, Jacquelyn Fetrow
States
POST 08-402 Bioinformatics and Network Analysis of
Lipocalin Superfamily Nardos Sori, Lesley H. Greene
POST 08-403 A New Evolutionary Subclass of HMGCoA Reductases Jeffrey Watson, James Palmer
POST 08-404 Ebola protein VP35 exploits
evolutionary constraints in host defense kinase PKR
to evade translational blockade Maayan Eitan-Wexler,
Raymond Kaempfer
POST 08-405 The substrate specificity “lock:”
evolutionary epistasis in Apicomplexan lactate and
malate dehydrogenases. Brian Beckett, Michelle Y. Fry,
Douglas L. Theobald
POST 08-406 Multi-level Iterative Functional
Clustering of Glutathione Transferase Superfamily
Kiran Kumar, Janelle Leuthaeuser, Brian Westwood,
Jacquelyn Fetrow
POST 08-407 Structural Analysis of Toxin/immunity
Complexes in Contact-Dependent Growth Inhibition
Systems Parker M. Johnson, Robert P. Morse, David A.
Low, Christopher S. Hayes, Celia W. Goulding
35
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 08-408 Automated Functional Clustering of the
Crotonase Superfamily based on active site motif
sequences Julia Hayden, Janelle Leuthaeuser, Brian
Westwood, Jacquelyn Fetrow
POST 08-409 Domain exchange between membrane
and soluble proteins contributes to expand
intercellular communication network Hyun Jun Nam,
Inhae Kim, James U. Bowie, Sanguk Kim
POST 08-410 Utilization of an Iterative Clustering
Method to group Radical SAMs in FunctionallyRelevant Ways Gabrielle B. Shea, Janelle B. Leuthauser,
Brian Westwood, Jacquelyn S. Fetrow
POST 08-411 Directed evolution of duplicated Qbeta
RNA replicases in liposome Keisuke Uno, Takeshi
Sunami, Yasuaki Kazuta, Norikazu Ichihashi, Tetsuya
Yomo
POST 08-412 Comparative Analysis of Posttranslational Modification Hot Spots in
Phylogenetically Distant Heterotrimeric G proteins
Shilpa Choudhury, Henry Dewhurst, Matthew P. Torres
Monday, 7/28/2014
POST 08-418 Synuclein and the Coelacanth
James M. Gruschus
Proteins in Altered States
POST 09-419 Storage of Environmental Information
into Biological Structures: Temperature-dependent
Fibrillar Polymorphism of α-Synuclein Ghibom Bhak,
Seung Ryeoul Paik
POST 09-420 Reversible Polymeric Fibers made from
Low-Complexity Sequences function as a Foundation
of RNA Metabolism Masato Kato, Steven McKnight
POST 09-421 Flavone derivatives as inhibitors of
insulin amyloid-like fibril formation Ricardas
Malisauskas, Akvile Botyriute, Vytautas Smirnovas
POST 09-422 Phosphorylation Releases Constraints to
Domain Motion in ERK2 Yao Xiao, Thomas Lee,
Michael P. Latham, Lisa R. Warner, Akiko Tanimoto,
Arthur Pardi, Natalie G. Ahn
POST 08-413 The evolution of caffeine synthases in
Theobroma cacao Craig D. Thulin
POST 09-423 Using mass spectrometry to define how
the small HSP chaperones protect substrates from
aggregation Keith Ballard, Heather O'Neill, Wenzhou
Li, Vicki Wysocki, Elizabeth Vierling
POST 08-414 Evolution in a test tube yields de novo
enzymes with unusual structure and dynamics
Burckhard Seelig, Frank A. Chao, Aleardo Morelli, John
C. Haugner, Lewis Churchfield, Gianluigi Veglia
POST 09-424 Mechanically resistant conformations in
amyloid β and α-synuclein Sigurdur Æ. Jònsson, Simon
Mitternacht, Anders Irbäck
POST 08-415 Quantitative Imaging with
Amersham’TM Imager 600 Erik Bjerneld, Erika
Svensson, Johan Johansson, Maria Winkvist, Hakan
Roos
POST 08-416 Solution NMR Structure of the SLED
Domain from Scml2 (Sex Comb on Midleg-like 2) - a
New Domain on Epigenetic Landscape. Irina
Bezsonova
POST 08-417 Monitoring the effect of extensive
genetic drift on the evolution of novel enzyme
function Linda Jäger, Peter Kast, Donald Hilvert
POST 09-425 Structural Determinants of Amyloid
Fibril Formation in Triosephosphate Isomerase.
Edson N. Carcamo-Noriega, Gloria Saab-Rincón
POST 09-426 Developing quantitative NMR methods
for predicting residue specific helicity of MetOλ
unfolded state Kan Li, Roy Hughes, Terrence G. Oas
POST 09-427 Towards in vivo NMR: structural studies
of prion fibrils assembled in native environments at
endogenous levels Kendra K. Frederick, Vladimir K.
Michaelis, Bjorn Corzilius, Ta-Chung Ong, Angela C.
Jacavone, Robert G. Griffin, Susan Lindquist
36
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 09-428 Structural and Ligand Binding Properties
of Dimeric Horse Myoglobin
Satoshi Nagao, Hisao Osuka, Takuya Yamada, Takeshi
Uni, Yasuhito Shomura, Kiyohiro Imai, Yoshiki Higuchi,
Shun Hirota
POST 09-429 Study of interactions of amyloidogenic
regions of Streptococcus mutans adhesin P1 by
Nuclear Magnetic Resonance Wenxing Tang, L.
Jeannine Brady, Joanna R. Long
POST 09-430 Identification and Characterization of
Functional Amyloids in Streptococcus mutans
Richard N. Besingi, L. Jeannine Brady
POST 09-431 Achieving selectivity in the Hippo
pathway: An investigation of the inter-domain
communication in TEAD transcription factors
Priyanka Rauniyar, Sudha Veeraraghavan
POST 09-432 Structural Projection of PTMs (SPoP): A
toolkit for providing structural and functional context
for sequence-specific protein features
Henry Dewhurst, Matthew P. Torres
POST 09-433 Investigating the Molecular Basis of
Curli Amyloid Inhibition by Protein and Chemical
Chaperones Neha Jain, Margery L. Evans, Matthew R.
Chapman
POST 09-434 The effects of mutations on the
aggregation propensity of human prion-like domains
Eric D. Ross, Kacy Paul, Sean Cascarina
POST 09-435 Competition or forced collaboration?
An unusual pattern of self-propagating polymorphism
of insulin amyloid fibrils upon seeding with mixed
templates. Wojciech Dzwolak, Weronika SurmaczChwedoruk
POST 09-436 Nanoscale Organization of Protein
Molecules within Amyloids and Prions Samrat
Mukhopadhyay
POST 09-437 A Temperature Sensitive Parkinsonian
Mutation in DJ-1 Enhances Protein Dynamics in a
Metal-dependent Fashion Nicole M. Milkovic, Steven
Monday, 7/28/2014
Halouska, Jonathan Catazaro, Sara Basiaga, Robert
Powers, Mark A. Wilson
POST 09-438 Analytical Tools in Decision Making of
Suitability of Monoclonal Antibodies for
Immunodiagnostic Assays Kevin R. Rupprecht, Tracey
D. Rae, Svetoslava D. Gregory, Na Yang, Janet M.
Bergsma, Ryan M. Bonn, Martin R. Lopez, Panfilo F.
Ozaeta, Cheng Zhao, Carol S. Ramsay, Jeffrey R.
Fishpaugh
POST 09-439 Spectroscopic Investigation of the
Structural Perturbations of Tau Microtubule Binding
Domains at the Golgi Membrane as Illustrated by
Membrane Mimics Lauren E. Sparks, Larry R.
Masterson
Proteins in Disease & Therapeutics
POST 10-50 Coupling the Antimicrobial Action of a
Novel Un-natural Cationic Amphiphilic Polyproline
Helix with its Cell Penetrating Ability to Target
Intracellular Bacteria Manish Nepal, Jean Chmielewski
POST 10-51 Rational design of the furin cleavage site
of an anti-CD22 recombinant immunotoxin based on
Pseudomonas exotoxin A John E. Weldon, Ira Pastan
POST 10-52 Structural and functional analysis of
lysosomal phospholipase A2, a close homolog of
lecithin-cholesterol acyltransferase Alisa Glukhova,
Robert J. Kelly, Vania Hinkovska-Galcheva, James A.
Shayman, John J. Tesmer
POST 10-53 Structural Basis for Antigen Recognition of
a Tumor Specific Therapeutic Antibody Reza
Movahedin, Teresa M. Brooks, Cory L. Brooks
POST 10-54 Neutralization of Listeria monocytogenes
by Single Domain Antibodies
Ian Huh, Robert Gene, Jyothi Kumaran, Cory Brooks
POST 10-55 Understanding regulation of P-Rex1, an
enhancer of metastatic potential Jennifer Cash, Ellen
Davis, John J. Tesmer
37
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 10-56 Expression and Purification of
Functionally Active Recombinant Plasmepsin 9 from
Plasmodium falciparum Folasade M. Olajuyigbe
POST 10-57 Characterizing HER2 gene variation at the
protein level to address racial disparities in breast
cancer mortality Wei He, Matthew Saldana, Tiffany
Scharadin, Steven Hoang-Phou, Denise Trans, Dennis
Chang, Kermit Carraway, Paul Henderson, Matthew A.
Coleman
POST 10-58 The interaction between the H. pylori
oncoprotein CagA and human pro-apoptotic ASPP2 is
distributed throughout both proteins and involves
intrinsically disordered regions Tali H. Reingewertz,
Anat Iosub-Amir, Daniel A. Bonsor, Assaf Friedler, Eric
J. Sundberg
POST 10-59 Is there a common structural basis for
amyloidosis toxicity? A new receptor-mediated
mechanism of pancreatic islet amyloidosis-induced
beta-cell toxicity in type 2 diabetes Andisheh Abedini,
Annette Plesner, Ping Cao, Jinghua Zhang, Fanling
Meng, Chris T. Middleton, Ling-Hsien Tu, Hui Wang, Fei
Song, Rosa Rosario, Martin T. Zanni, Bruce Verchere,
Daniel P. Raleigh, Ann Marie Schmidt
POST 10-60 SUMO4 C438T polymorphism is
associated with papulopustular skin lesion in Korean
patients with Behçet’s disease Hyun-Sook Kim
POST 10-61 Calcium-dependent proteases and their
proteinaceous inhibitor in the brain regions affected
by neurodegeneration in rats Liudmila A. Lysenko,
Nadezda P. Kantserova, Nikolay L. Rendakov, Khristina
N. Prokopenko, Nina N. Nemova
POST 10-62 Monomeric IKK: Probing Activation and
Specificty William E. Rogers
POST 10-63 An Ultrasensitive platform for the
detection of protein biomarkers in spiked human
serum Stephen Vance, Marinella Sandros
POST 10-64 Regulation of calpain activity in fish brain
by weak low-frequency magnetic fields
Monday, 7/28/2014
Nadezda P. Kantserova, Liudmila A. Lysenko, Natalia V.
Ushakova, Vyacheslav V. Krylov, Nina N. Nemova
POST 10-65 Amyloid beta peptide Aβ40 and Aβ42
form separate fibrils in binary mixtures Xiaoting Yang,
Risto Cukalevski, Georg Meisl, Birgitta Frohm, Tuomas
Knowles, Sara Linse
POST 10-66 Molecular engineering of L-asparaginases
as therapeutic enzymes for the treatment of leukemia
Manfred W. Konrad, Christos S. Karamitros
POST 10-67 Tweaking the spines of Kinase Structures
Lalima G. Ahuja, Jiancheng Hu, Alexandr P. Kornev,
Andrey S. Shaw, Susan S. Taylor
POST 10-68 Correlation between Aβ (1-40)
aggregation in E.coli and in vitro amyloid fibril
formation Kalyani Sanagavarapu, Irem Nasir, Celia
Cabaleiro-Lago, Sara Linse
POST 10-69 Structure-based design of dual smallmolecule inhibitors of Mdm2/MdmX for efficiently
reactivating p53 in cancer cells Zhengding Su, David
Duda, Lingyun Qin, Yao Chen, Huashan Zhang, Weiping
Wang, Brenda Schulman
POST 10-70 Subtle Differences Between NOS Active
Sites Lends Towards the Development of a Bacterial
NOS Specific Inhibitor Jeffrey K. Holden, Soosung Kang,
Richard B. Silverman, Thomas L. Poulos
POST 10-71 The role of extracellular EMMPRIN and its
glycosylation in modulating cancerous phenotypes
Agnieszka A. Kendrick, Elan Z. Eisenmesser
POST 10-72 Structural insights into the role of the
Smoothened cysteine rich domain in Hedgehog
signalling Rajashree Rana, Candace Carroll, Ho-Jin Lee,
Ju Bao, Suresh Marada, Grace Royappa, Stacey Ogden,
Jie Zheng
POST 10-73 CRAMP 16-33 inhibits the assembly and
GTPase activity of FtsZ and perturbs the formation of
the cytokinetic Z-ring in bacteria
Shashikant Ray, Dulal Panda
38
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 10-74 Dimerized Translationally Controlled
Tumor Protein (TCTP) induces Interleukin-8 secretion
in human bronchial epithelial cells via MAPK and NFκB pathway. Heewon Lee, Haejun Pyun, Jeehye
Maeng, Kyunglim Lee†
POST 10-75 Lysolipids Modulate Aggregation of the
Repeat Domain of a Human Functional Amyloid,
Pmel17 Zhiping Jiang, Jennifer C. Lee
POST 10-76 Extracellular clusterin suppresses the
formation of cytotoxic -α-synuclein species by
interacting with prefibrillar species and facilitates
their lysosomal degradation: Implications in
Parkinson’s disease Abdullah Sultan, Bakthisaran
Raman, Ch M. Rao, Ramakrishna Tangirala
POST 10-77 The adhesion regions of gingipains from
P. gingivalis are composed of at least two distinct
types of domains . Charles A. Collyer, Jinlong Gao,
Daniele Vicari, Nan Li
POST 10-78 Alzheimer’s disease and cerebral amyloid
angiopathy, doppelgangers? Rabia Sarroukh, Ellen
Hubin, Louise C. Serpell, Nico A. van Nuland, Kerensa
Broersen, Vincent Raussens
POST 10-79 Investigating of the immunomodulatory
function of Plasmodium falciparum Hsp70 expressed
from various LPS minus bacterial strains Ofentse J.
Pooe, Gabriele Köllisch, Holger Heine, Addmore
Shonhai
POST 10-80 Near infrared spectral monitoring reveals
water molecular system dynamics during the
amyloidogenic nucleation Eri Chatani, Yutaro
Tsuchisaka, Yuuki Masuda, Roumiana Tsenkova
POST 10-81 Liver Internalization of Cholesterol
Decoded by X-rays Hay Dvir
POST 10-82 RNA structural elements and protein
interactions that regulate HIV genome splicing
Blanton S. Tolbert
POST 10-83 Molecular mechanism by which an
intrinsically disordered region in non-segmented
Monday, 7/28/2014
negative strand RNA virus phosphoprotein acts as a
chaperone of unassembled viral nucleoprotein
Cédric Leyrat, Filip Yabukarski, Malene R. Jensen, Rob
Ruigrok, Martin Blackledge, Marc Jamin
POST 10-84 Substrate and drug induced
conformational heterogeneity in CETP Revathi Sankar,
Sanjib Senapati
POST 10-85 ASB9 N-terminus plays an important role
in creatine kinase regulation Deepa Balasubramaniam,
Jamie Schiffer, Jonathan Parnell, Stephan Mir, Elizabeth
Komives
POST 10-86 Limited proteolysis and dissociation of
trimeric state of the BRICHOS domain increase its
anti-amyloid activity Henrik Biverstål, Lisa Dolfe, Erik
Hermansson, Jenny Presto, Jan Johansson
POST 10-87 Thermodynamic and structural
characterization of the binding of Zn(II) and other
molecules to human protein DJ-1 Shinya Tashiro, Jose
Caaveiro, Chun-Xiang Wu, Quyen Hoang, Kouhei
Tsumoto
POST 10-88 PICH and BEND3 form a complex:
potential role in the processing of ultrafine anaphase
DNA bridges. Ganesha p. Pitchai
POST 10-89 Computational Docking and Site Directed
Mutagenesis to Identify Steroid Binding Sites on
Ionotropic Glutamate Receptors Emily Bartle, Philip
Varnes, Carol Parish, Ellis Bell
POST 10-90 Effects of Agonist and Regulator Binding
on the Structure and Conformational Flexibility of the
Ligand Binding Domains of Ionotropic Glutamate
Receptors Forest Barkdoll-Weil, Carol Guzman, Philip
Varnes, Ellis Bell
POST 10-91 A Novel CD4-based Chimeric Antigen
Receptor for Functional Cure of HIV Li Liu, Bhavik
Patel, Mustafa Ghanem, Zhilli Zheng, Virgilio Bundoc,
Richard Morgan, Steven A. Rosenberg, Barna Dey,
Edward A. Berger
39
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 10-92 Some Like it Hot: Determination of
Biomolecular Interactions using MicroScale
Thermophoresis Ana Lazic, Wyatt Strutz, Nicole Ford,
Stefan Duhr
POST 10-93 Effects of Regulator Binding on the
Structure and Conformational Flexibility of the Amino
Terminal Domains of Ionotropic Glutamate Receptors
Carlos Metz, Emily Bartle, Alaina Hyde, Ellis Bell
POST 10-94 The variant of CREBBP with HAT domain
deletion inhibits Hela cell proliferation by downregulating the expression of TAF1 Zhang Lu-yu, Li Xin,
Wang Yun-hong, Wen Quan, Zhang Jun
POST 10-95 Effect of Polyethylene Glycol Conjugation
on Conformational and Colloidal Stability of a
Monoclonal Antibody Antigen Binding Fragment (Fab)
Cristopher Roque, Anthony Sheung, Nausheen
Rahman, Salvador F. Ausar
POST 10-96 Biochemical and structural
characterization of LiaR from Vancomycin-Resistant
E. faecalis : the ‘master regulator’ of the cell-envelope
stress response . Milya Davlieva, Yiwen Shi, Michael
Zianni, Troy Johnson, Paul Leonard, John Ladbury,
Cesar A. Arias, Yousif Shamoo
POST 10-97 Potent inhibition of α-synuclein
fibrillization and toxicity through an energyindependent chaperone-like activity Jan Bieschke
POST 10-98 Rational search for a compound that
selectively inhibits the triose phosphate isomerase
from Trichomonas vaginalis José L. Vique, Luis G.
Brieba, Rossana Arroyo, Jaime Ortega, Arturo Rojo,
Ponciano Garcia, Claudia Benítez
POST 10-99 The level of Dot1L recruitment defines the
degree of MLL-AF9 hematopoietic transformation
Aravinda Kuntimaddi, John Bushweller
POST 10-100 Long-range Activity Regulation
Mechanisms Within The IL-33/ST2/IL-1RAcp Complex
Kendra Hailey, Bryan E. Jones, Patricia A. Jennings
Monday, 7/28/2014
POST 10-101 Modeling and Simulation of Full-length
p53 Tetramer Bound to DNA Ozlem Demir, Pek Ieong
POST 10-102 Putative programmed cell death
pathway of the malaria parasite and the role of
cytochrome C Judith H. Prieto
POST 10-103 Misfolding of the vWF A1 Domain alters
the strength of platelet adhesion in Type 2 von
Willebrand Disease Alexander Tischer, Pranathi
Madde, Laurie Moon-Tasson, Matthew Auton
POST 10-104 Predictive and experimental approaches
for characterizing mutations in proteins
Maria Teresa Buenavista, Rohanah Hussain, Ann-Marie
Mallon, David Nutt, Liam James McGuffin
POST 10-105 Structural unity in diversity in pilins of
some enteric pathogens Himadri Biswas, Rajagopal
Chattopadhyaya
POST 10-106 Synthesis of selectively functionalized
adiponectin Andreas Mattern, Annette G. BeckSickinger
POST 10-107 Structural analysis of the CFA/III minor
pilin subunit CofB of human enterotoxigenic
Escherichia coli Hiroya Oki, Kazuki Kawahara, Shunsuke
Fukakusa, Takuya Yoshida, Yuji Kobayashi, Tooru
Taniguchi, Takeshi Honda, Tetsuya Iida, Shota
Nakamura, Tadayasu Ohkubo
POST 10-108 Analysis of the gene products related to
osseointegration in the early stage of titanium
implantation Masataka Horiuchi, Rumi Horiuchi,
Masanori Ochiai, Atsuro Yokoyama
POST 10-109 Advanced Molecular Tools for
Proteomic Analyses of Microvesicles Masood KamaliMoghaddam, Liza Löf, Felipe Oliveir, Lotta Wik, Di Wu,
Junhong Yan
POST 10-110 Bovine Brain Ribonuclease is the
Functional Homolog of Human Ribonuclease 1
Chelcie H. Eller, Jo E. Lomax, Ronald T. Raines
40
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 10-111 Defect in the Intramolecular and
Intermolecular Cross-linking of Collagen Caused by
Hcy-thiolactone Marta Rusek
POST 10-112 Thermodynamic Analysis of Membrane
Interactions with Tau Peptides Megan Culp, Larry
Masterson
POST 10-113 Molecular basis of heme capture by Isd
system of Staphylococcus aureus Jose M. Caaveiro,
Nhuan Vu, Koldo Morante, Yoshitaka Moriwaki, Ryota
Abe, Kouhei Tsumoto
POST 10-114 Biologically responsive recombinant
protein anchors for macromolecular drug delivery
Jason S. Buhrman, Jamie E. Rayahin, Yu Zhang, Mary
Tang, Richard A. Gemeinhart
POST 10-115 X-ray crystallography & Small Angle Xray Scattering studies of Interferon Regulatory Factor
4 Soumya Govinda Remesh, Carlos R. Escalante
POST 10-116 Structural Characterization of Protein
Aggregates of Wild-Type and Disease Associated
Variants of Human γS-crystallin David M. Montelongo,
Chelsea Anorma, Diana Bandak, Rachel W. Martin
POST 10-117 Insight into the Catalytic Mechanism of
GABA - producing Enzyme: Glutamate Decarboxylase
from Sphaerobacter thermophilus
Ruiying Wu, Shonda Clancy, Andrzej Joachimiak
POST 10-118 Interactions of E. coli immunoglobulin
binding protein D and the Fc part of IgG Kornelia M.
Mikula, Robert Kolodziejczyk, Adrian Goldman
POST 10-119 The Dynamic Functional Consequences
of the Thrombin-Thrombomodulin Interaction
Lindsey D. Handley, Elizabeth A. Komives
POST 10-120 A Novel Activator of ATPase Activity of
NBD1 Domain of the CFTR Jay Singh, William Balch
POST 10-121 Targeting the NEET Proteins for Cancer
Treatment Colin H. Lipper, Mark L. Paddock, José N.
Onuchic, Ron Mittler, Rachel Nechushtai, Emmanuel
A. Theodorakis, Patricia A. Jennings
Monday, 7/28/2014
POST 10-122 Understanding the role of human DDX21
RNA helicase in HIV-1 Rev-RNA assembly in vitro Li
Zhou, James R. Williamson
Proteins in Dynamic & Driven
Processes
POST 11-123 Characterization of a temperature
responsive two-component regulatory system in the
Antarctic methanogen, Methanococcoides burtonii
POST 11-124 Crystal Structure of the Periplasmic
Sensor Domain of Histidine Kinase CusS Bound to
Silver
Trisiani Affandi, Aaron V. Issaian, Sue A. Roberts,
Megan M. McEvoy
POST 11-125 Structural Investigation into the
Mechanism of the Synthase Subunit of PLPS. Amber
M. Smith, Janet L. Smith
POST 11-126 Super Spy variants implicate flexibility in
chaperone action Shu Quan, Lili Wang, Evgeniy V.
Petrotchenko, Karl A. Makepeace, Scott Horowitz,
Jianyi Yang, Yang Zhang, Christoph H. Borchers, James
C. Bardwell
POST 11-127 Elucidation of the nonspecific DNAbinding mechanism of the POU homeodomain using
NMR Tsuyoshi Konuma, Erisa Harada, Takashi Oda,
Mamoru Sato, Kenji Sugase
POST 11-128 Time-lapsing Planaria: Studying
Planarian regeneration by stop-motion imaging and
by pulse-isotopic proteome labeling Wei Shen,
Karsten Berning
POST 11-129 Hydrogen Deuterium Exchange Mass
Spectrometry and Molecular Dynamics Reveal the
Interactions of Membrane Phospholipids and
Inhibitors with Phospholipases A2 Varnavas Mouchlis,
Denis Bucher, J. Andrew McCammon, Edward A.
Dennis
41
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 11-130 Visualizing the inter-domain motions of
a flexible protein using continuous models
Yang Qi, Jeffrey W. Martin, Bruce R. Donald, Terrence
G. Oas
POST 11-131 Benchmarking FRET for live-cell PPI
Tao Lin, Brandon Scott, Francisca Essel, Moul Dey,
Adam Hoppe, Suvobrata Chakravarty
POST 11-132 Dynamics and chaperone function in the
small heat-shock protein αB-crystallin Georg
Hochberg, Heath Ecroyd, Dezerea Cox, Michael
Sawaya, Cong Liu, Duilio Cascio, Miranda Collier, James
Stroud, John Carver, Andrew Baldwin, Carol Robinson,
David Eisenberg, Justin Benesch, Arthur Laganowsky
Monday, 7/28/2014
POST 11-140 Intrinsic GTP Hydrolysis is Observed For
a Switch 1 Mutant of Cdc42 in the Presence of a
Specific GTPase Inhibitor Reena Chandrashekar, Kyla
Morris, Colin D. Heyes, Paul D. Adams
POST 11-141 Three-dimensional Structure of the 54
kDa Subunit of the Chloroplast Signal Recognition
Particle using Molecular Modeling Rory Henderson,
Suresh Kumar, Colin Heyes, Ralph Henry
POST 11-142 Evidence for significant changes in
backbone motions between apo and Gα-bound
human RGS4 Lusine Simonyan, Shayla A. Brooks, Karin
A. Crowhurst
POST 11-133 Protein Flexibility and Gymnastics Drive
Robust Clockwise Ticking of a three-protein KaiABC
Oscillator Yonggang Chang, Roger Tseng, Andy LiWang
Protein Engineering & Synthetic
Biology
POST 11-134 Characterization of Dynamic processes in
substrate recognition by cytochrome P450 enzymes
Nitin Jain, Nicholas Lopes, Ana Bernal
POST 12-143 Engineering Picomolar Affinity into a
Rationally Identified 5 kDa Scaffold for Tumor
Targeting Max Kruziki, Patrick Holec, Benjamin Hackel
POST 11-135 Mapping the Interactions between the
Molecular Chaperones Hsp70, Hsp104 and Hsp110
Shankar Shastry, Shannon Doyle, Joel Hoskins, Sue
Wickner
POST 12-144 Utilizing Metal-Ligand Interactions to
Promote Assembly of Collagen-Based Peptides into
Functional Nanostructures Jeremy Gleaton, David
Przybyla, Charles M. Rubert-Perez, Jean Chmielewski
POST 11-136 The role of Phenylalanine in an
intrinsically disordered protein from yeast
nucleoporins. Korey M. Reid, Krish Krishnan
POST 12-145 Live Cell Imaging of Molecular
Conformations and Actions Peter Yingxiao Wang
POST 11-137 The Acidic Residues of the IκBα PEST
Sequence are Responsible for Actively Dissociating
NFκB from DNA Holly E. Dembinski, Kevin Wismer,
Elizabeth Komives
POST 11-138 Nanoscale Hydrodynamic Study of
Proteins under Thermal Agitation and Electric Field
Yuanming Zhang, Zachary Weiner, Eric Farrell
POST 11-139 Probing the clamping movement of
xylanase B by NMR spectroscopy Nhung T. Nguyen,
Nicolas Doucet
POST 12-146 Protein design: Preventing protein
aggregation in recombinant erythropoietin
Manuel A. Carballo-Amador, Jim Warwicker, Alan J.
Dickson
POST 12-147 Intracellular regulation of the NFE2L3
transcription factor Meenakshi B. Kannan, Volker
Blank
POST 12-148 Phosphorylation and Binding Partners of
the NFE2L3 Transcription Factor: Insights into its Role
in Oncogenesis Isadore Dodard-Friedman, Volker Blank
42
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 12-149 An Orthogonal Genetic System for Rapid
Evolution Chang Liu
POST 12-150 A recognition model of ACP-HCS
interaction for programmed beta-branching in type I
polyketide synthases Rohit Farmer, Anthony S. Haines,
Matthew Crump, Christopher Thomas, Peter J. Winn
POST 12-151 Engineering photo-control of translation
initiation using photoactive yellow protein hybrids
Anil Kumar, Anna S. Jaikaran, Alaji Bah, Julie FormanKay, G. Andrew Woolley
POST 12-152 Gold Decorated Peptide Amphiphile
Templates for Directed Silver Nanorods Growth
Shlomo Zarzhitsky, Hanna Rapaport
POST 12-153 Engineered oligosaccharyltransferases
with greatly relaxed acceptor site specificity Anne A.
Ollis, Sheng Zhang, Matthew P. DeLisa
POST 12-154 The dynamic peptide recognition and
stabilization mechanism of Human Leukocyte Antigen
B*35:01 Saeko Yanaka, Takamasa Ueno, Kouhei
Tsumoto, Kenji Sugase
POST 12-155 Engineering Ordered Protein Assemblies
by Helix-Fusion Strategy Yen-Ting Lai, Todd O. Yeates
POST 12-156 Total synthesis and chaperonemediated folding of a 312-residue mirror-image
enzyme Michael T. Jacobsen, Matthew T. Weinstock,
Michael S. Kay
POST 12-157 Creating self-assembling stimulusresponsive hydrogels from protein components
Danielle Williams, Ashley Schloss, Lynne Regan
POST 12-158 Characterization of a novel synthetic
biomaterial for protein immobilization Carrie MareanReardon, Patrick Reardon, Thomas Squier, Kathleen
McAteer
POST 12-159 Protein Fragment Exchange: Converting
an Arbitrary Binding Protein into a Robust FRET
Biosensor Huimei Zheng, Jing Bi, Mira Krendel, Stewart
N. Loh
Monday, 7/28/2014
POST 12-160 Design of an Albumin-Binding Human
Protein by Mimicking the Contact Surface of a
Bacterial Albumin-Binding Domain Satoshi Oshiro,
Shinya Honda
POST 12-161 Developing soluble co-receptor mimetics
for the study of HIV Env/receptor interactions Agnes
Hajduczki, Virgilio Bundoc, Edward A. Berger
POST 12-162 Constructing highly detectable
fluorescence reporter protein for in vitro single
molecular screening Kotaro Nishiyama, Norikazu
Ichihashi, Yasuaki Kazuta, Tetsuya Yomo
POST 12-163 Model building of antibody-antigen
complex structures using GB/SA scores Narutoshi
Kamiya, Noriko Shimba, Haruki Nakamura
POST 12-164 Engineering Novel Phosphopeptide
Recognition Modules that Recognize Targets In Vitro
and in E. coli Nicholas Sawyer, Lynne Regan, Brandon
Gassaway, Jesse Rinehart, Adrian Haimovich, Farren
Isaacs
POST 12-165 N-linked glycosylation of HIV-1 core
gp120 is not required for native trimer formation or
viral infectivity Ujjwal Rathore, Piyali Saha, Sannula
Kesavardhana, Aditya A. Kumar, John R. Mascola,
Raghavan Varadarajan
POST 12-166 Silicon transporters: from membrane
proteins to nanotechnology Laura Senior, Sarah
Ratcliffe, Michael Knight, Adam Perriman, Stephen
Mann, Paul Curnow
POST 12-167 Characterization of Thermotoga
maritima Maltotriose Binding Protein Laura Masson,
Jonathan Dattelbaum
POST 12-168 Zinc induces self-assembly of bacterial
thermoalkalophilic lipases: a strategy for
thermostability Emel Timucin, Osman U. Sezerman
POST 12-169 The SasG E-G5 protein fold forms a
stable contiguous rigid nanorod of tunable length
Fiona Whelan, Dominika T. Gruszka, Jane Clarke,
Jennifer R. Potts
43
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 12-170 Computational design of allosteric
antibody Olga Khersonsky, Sarel Fleishman
POST 12-171 Development and optimization of an E.
coli -based display platform for selection of affinity
proteins Filippa Fleetwood, Ken Andersson, Stefan
Ståhl, John Löfblom
Monday, 7/28/2014
POST 12-181 Beta-hairpin tags for increased
expression of helical peptides Melissa E. Lokensgard,
John J. Love
POST 12-182 Gradient Diversity Enriches
Combinatorial Protein Library Design Daniel R.
Woldring, Benjamin J. Hackel
POST 12-173 Accurate prediction of protein stability
by explicit negative multistate design James A. Davey,
Christian K. Euler, Roberto A. Chica
POST 12-183 Expanding the GFP toolbox through a
better understanding of its folding pathway Keith
Fraser, Colleen Lamberson, Victoria Jones, Erin Gilbert,
David Rosenman, Yao-ming Huang, Derek Pitman,
Shounak Banerjee, Yan Xia, Angela Choi, Rachel
Altshuler, Luis Garreta, John Karonicolas, Donna E.
Crone, Jonathan S. Dordick, Christopher Bystroff
POST 12-174 Plastic protein design: A novel design
algorithm using backbone and side-chain ensembles
to model protein flexibility Christian D. Schenkelberg,
Derek J. Pitman, Yao-ming Huang, Christopher Bystroff
POST 12-184 Are Peptide Nucleic Acids (PNAs)
Recognized by Aminoacyl-tRNA Synthetases (aaRSs)?
Crystal Serrano, Long Nguyen, Anthony Bell, Filbert
Totsingan
POST 12-175 Repeat-protein directed synthesis of
gold nanoparticles with tunable morphology and
optical properties Tijana Grove, Xi Geng
POST 12-185 Protein Rings and Tubes as Versatile
Templates for Self-Assembled Bionano Structures
Ali A. Malay, Zuben Brown, Kenji Iwasaki, Jonathan G.
Heddle
POST 12-172 Computational Redesign of
Metagenomic Enzymes Justin B. Siegel
POST 12-176 Designed Affinity Reagents Directed to
Heat Shock Protein C-terminal Motifs Robert Wells,
Akiko Koide, Shohei Koide
POST 12-177 Consensus design of a NOD receptor
leucine rich repeat domain with binding affinity for a
muramyl dipeptide (MDP), a bacterial cell wall
fragment Rachael Parker, Ana Mercedes-Camacho,
Tijana Z. Grove
POST 12-178 Solvent engineering to improve
enzymatic synthesis of a sugar-based surfactant
Rodrigo A. Arreola-Barroso, Gloria Saab-Rincón
POST 12-179 Delivery of natural and non-natural
chemical entities into cells using anthrax toxin
Amy Rabideau, Xiaoli Liao, Bradley Pentelute
POST 12-180 Thermostabilization of Aspergillus
oryzae Cutinase Danielle A. Basore, Abhijit Shirke,
Glen Butterfoss, Richard Gross, Christopher Bystroff
POST 12-186 BIO-INSPIRED TECTONS: The
architecture & engineering of synthetic ring-forming
proteins Francesca Manea, Bridget C. Mabbutt
POST 12-187 A designed small protein for controlling
site-specific mineralization of silica and calcium on
DNAs Kenji Usui, Kazuma Nagai, Hiroto Nishiyama, Aoi
Yamada, Makoto Ozaki, Takaaki Tsuruoka, Kin-ya
Tomizaki
POST 12-188 Enhancing predictions of surface entropy
reduction for improved crystallizability of proteins
Derek J. Pitman, Thomas Holton, Luki Goldschmidt,
Zygmunt Derewenda, David Eisenberg
POST 12-189 Repeat protein scaffolds for assembly of
functional nanostructures Sara H. Mejias, Pierre
Couleaud, Javier Lopez, Begoña Sot, Carmen Atienza,
Teresa Gonzalez, Aitziber L. Cortajarena
44
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 12-190 Generation of Novel Amino Acid
Dehydrogenase Activity through Evolution of a Highly
Specific Alanine Dehydrogenase Emily Mundorff
POST 12-191 Rational protein design, SPR binding
analysis, and iterative optimization of studentdesigned single-chain immunoreceptors Benjamin J.
McFarland, Andrew Daman
Monday, 7/28/2014
POST 12-200 Protein-based stimuli-responsive
hydrogels for targeted drug delivery Ashley C.
Schloss, Abbie Omolu, Richard Day, Lynne J. Regan
POST 12-201 β/α and α/β of TIM Barrel Proteins:
Database and Structural Analysis Ramakrishna
Vadrevu, Rajashekar V. Kadumoori
POST 12-192 Efficient gene disruption at hTERT
promoter region by simultaneous digestion by pairs of
ZFNs or guide RNAs for CRISPR/Cas system.
Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
POST 12-202 De Novo Design of Peptides that
Assemble Lipid Nanodiscs Shao-Qing Zhang, Kazuma
Yasuhara, Hyunil Jo, William F. DeGrado
POST 12-193 High-throughput sorting of the highest
producing cell via a transiently protein-anchored
system Kuo-Hsiang Chuang
POST 12-194 Dissecting contributions to protein
stability via recombination of a wild type and
computationally designed protein Lucas Johnson,
Lucas Gintner, Luke Minardi, Christopher Snow
POST 12-203 A Hyperstable Minimalist Protein for
Molecular Recognition Michael W. Traxlmayr, Raja R.
Srinivas, Elisabeth Lobner, Bruce Tidor, Karl D. Wittrup
POST 12-195 Computational Design of Protein Ligand
Interfaces Using RosettaLigand Brittany Allison, Jens
Meiler
POST 12-205 “PA tag”, a versatile affinity tag system
that enables one-step affinity purification and high
sensitive detection of recombinant proteins from
dilute sample Yuki Fujii
POST 12-196 Eliminating Endotoxin at the Source - A
Novel Competent Cell Line with Modified
Lipopolysaccharide for Low-Endotoxin Plasmid
Production Saurabh Sen, Uwe Mamat, Chad
Souvignier, Eric Steinmetz, Chelsea Kovacich, David
Mead, Curtis Knox
POST 12-197 Computational Design of Leucine-Rich
Repeat- based Protein Binding Scaffolds. Sebastian
Rämisch, Ulrich Weininger, Ingemar André
POST 12-204 A Synthetic Biochemistry Molecular
Purge Valve Module that Maintains Redox Balance
Paul Opgenorth
POST 12-206 Computational design of protein-DNA
nanowires Yun Mou
POST 12-207 Reversible modification of the Nterminal cysteine residue of proteins using pyruvic
acid analogs Pradeep Budhathoki, Youngha Ryu
POST 12-208 AbDesign : Computational antibody
design Switching species preference and humanizing
an inhibitory antibody Assaf Alon, Sarel J. Fleishman
POST 12-198 Generation and Characterization of the
Native-like IgG Bispecific Antibodies Steven M.
Lewis, Arlene Sereno, Flora Huang, Anna Pustilnik,
Heather L. Rick, Elaine M. Conner, Shane Atwell, Brian
Kuhlman, Stephen J. Demarest, Xiuifeng Wu
POST 12-209 Sequence-specific cleavage of initiating
methionine and RimJ-catalyzed N-terminal
acetylation of the Z-domain in Escherichia coli
Youngha Ryu, Lina Bernal-Perez
POST 12-199 Improving the efficiency of
Concanavalin A as affinity ligand in alkaline pH range
Akash Chaudhary, Shakeel Ahmad, Shadab Ahmad,
Mohd. Tashfeen Ashraf
POST 12-210 In vitro selection and evolution of
membrane proteins using liposome display Tomoaki
Matsuura, Satoshi Fujii, Yasuaki Kazuta, Takeshi
Sunami, Tetsuya Yomo
45
POSTER SESSIONS
Grand Hall A & B
Sunday, 07/27/2014
POST 12-211 Ubiquibodies: Engineered E3 Ubiquitin
Ligases for Targeted Degradation Erin A. Stephens,
Alyse D. Portnoff, Morgan R. Baltz, Jeffrey D. Varner,
Matthew P. DeLisa
POST 12-212 Engineering Ubiquitin to recognize noncognate proteins implicated in cancer Isabel Leung,
Sachdev Sidhu, Nick Jarvik
POST 12-213 Optimization of a protein labelling
technique for fluorogenic, X-ray crystallography and
NMR applications Miroslava Strmiskova, Natalie K.
Goto, Jeffrey W. Keillor
Monday, 7/28/2014
POST 12-221 Beyond Glutaraldehyde: The Search for
Optimal Chemical Crosslinkers for Protein Crystal
Thaddaus R. Huber, Jacob Sebesta, Christopher Snow
POST 12-222 Utilization of Collagen IV NC1 Domains
to Control Helical Composition: A Recombinant
Strategy for the Production of Collagen IV Protomers
Kyle L. Brown, Vadim Pedchenko, Selene Colon, Tim
Blackwell, Ambra Pozzi, Billy Hudson
POST 12-223 Secondary Antibody Cross-Reactivity
Identification Using ProteOn XPR36 System Gary
Ross, Perry Ripa, Mohammed Yousef
POST 12-214 Computational Design of Cystatin and
Ketosteroid Isomerase Folds from Scratch: From
Structure to Self-Labeling Function Enrique Marcos,
Dr. David Baker
POST 12-215 Development of Novel p16INK4a
Peptide Mimetics as Anticancer Therapy Marian
Kratzke, Yuk Sham, Mark A. Klein
POST 12-216 A new protein cage architecture formed
via gold cluster catalysis Ali D. Malay, Kenji Iwasaki,
Zuben Brown, Jonathan G. Heddle
POST 12-217 Identification of residues in TIMP-1 that
are critical in interaction between MMP-9 and TIMP1. Ruiying Wang, Alexandra Hockla, Evette S. Radisky
POST 12-218 Computationally Designed Green
Fluorescent Protein Based Biosensors Shounak
Banerjee, Yao-Ming Huang, Donna E. Crone, Diana I.
Paredes, Jonathan S. Dordick, Christopher Bystroff
POST 12-219 Atomic Force Microscopy
Characterization of Beta-Solenoid Based Amyloid
Fibrils Arpad Karsai, Maria D.R. Peralta, Alice Ngo, N.
Robert Hayre, Nima Mirzaee, Alexander J. Kluber, Xi
Chen, Gang-yu Liu, Michael Toney, Rajiv R.P. Singh,
Daniel L. Cox
POST 12-220 Reverse Protein Engineering towards a
fluorescent peptide Zhiwen J. Zhang, Blake Williams
46
EXHIBITOR DIRECTORY
Anton Paar USA Booth # 103
10215 Timber Ridge Drive
Ashland, VA 23005
Phone
Fax
Email
Web
804-550-1051
804-550-1057
[email protected]
http://www.anton-paar.com
SAXSpace is a laboratory instrument for small-angle and wide-angle X-ray scattering (SAXS and
WAXS/XRD). Combined in the SAXSpace system, these two techniques form a powerful
analytical solution for investigating liquid crystals, dispersions, polymers, proteins, and much
more. A compact instrument that provides ultimate clarity for your materials research and
quality control.
AVIV Biomedical, Inc.
Booth #204
750 Vassar Avenue, Suite 2
Lakewood, NJ 08701-6929
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Fax
Email
Web
732-370-1300
732-370-1303
[email protected]
www.avivbiomedical.com
Aviv Biomedical Inc. manufactures scientific and clinical instruments. Products include a
fluorescence accessory (AU-FDS) for the Beckman Analytical Ultracentrifuge, model XLA/XLI.
Sales, service and support of Aviv Spectrometers, Aviv Spectrophotometers and Aviv
Fluorometers.
BioLegend
Booth # 210
9727 Pacific Heights Blvd
San Diego, CA 92121
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858-455-9588
877-455-9587
[email protected]
www.biolegend.com
World-Class Antibodies, Proteins, Assays and Research Solutions. Complete Brilliant Violet™
Antibody Conjugates for the Violet Laser: BV510™, BV711™, BV785™. Personalized Multicolor
Flow Cytometry Panel Design. New LEGENDScreen™ Human Cell Screening (PE) Kits. Request
Bulk Cytokines & Chemokines for Bioassay. Ultra-LEAF™ (Low Endotoxin, Azide-Free)
Antibodies. New ELISA Kits: IL-35, Active TGF- β1.
47
EXHIBITOR DIRECTORY
Brookhaven Instruments Corporation
750 Blue Point Road
Holtsville, NY 11742
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Email
Web
Booth # 212
631-758-3200
631-758-3255
[email protected]
www.BrookhavenInstruments.com
Brookhaven pioneered modern techniques in characterizing nanoparticles, proteins, and
polymers using light scattering for particle sizing, zeta potential, and absolute molecular weight
analysis. New SEC/GPC software, ParSEC, works with almost any detector on the market. New
particle characterization software, Particle Solutions, is a unique, database approach with
unsurpassed search features.
Cayman Chemical Company
1180 East Ellsworth Rd
Ann Arbor, MI, 48108
Booth # 105
Phone 734 971-3335
Fax 734 971-3420
www.caymanchem.com
Our scientists are experts in the synthesis, purification, and characterization of biochemicals
ranging from small drug-like heterocycles to complex biolipids, fatty acids, and many others. We
are also highly skilled in all aspects of assay and antibody development, protein expression,
crystallization, and structure determination. Our organic and analytical chemists specialize in
the rapid development of manufacturing processes and analytical methods to carry out clinical
and commercial GMP-API production. Pre-clinical drug discovery efforts are currently
underway in the areas of bone restoration and repair, muscular dystrophy, oncology, and
inflammation. A separate group of Ph.D.-level scientists are dedicated to offering Hit-to-Lead
Discovery and Profiling Services for epigenetic targets. Our knowledgeable chemists can be
contracted to perform complete sample analysis for analytes measured by the majority of our
assays. We also offer a wide range of analytical services using LC-MS/MS, HPLC, GC, and many
other techniques. Cayman is a leader in the field of emerging drugs of abuse, providing highpurity Schedule I-V Controlled Substances to federally-licensed laboratories and qualified
academic research institutions for forensic analyses. We are certified by ACLASS Accreditation
Services with dual accreditation to ISO/IEC 17025:2005 and ISO Guide 34:2009.
FASEB MARC Program
9650 Rockville Pike
Bethesda, MD 20814
Phone
Fax
Email
Web
Booth #205
301-634-7930
301-634-7353
[email protected]
www.Faseb.org/marc
48
EXHIBITOR DIRECTORY
The FASEC MARC (Maximizing Access to Research Careers) Program provides a variety of
activities to support the training of students, postdoctorates, faculty and researchers from
underrepresented groups who are engaged in the biomedical and behavioral sciences research
and training. We offer faculty/mentor with students and poster/platform presenter travel
awards for scientific meetings (national and regional) and FASEB Science Research
Conferences. We also sponsor career/leadership development and grantsmanship training
seminars and workshops.
GE Healthcare
Booth # 221
800 Centennial Avenue – PO Box 1327
Piscataway, NJ 08855-1327
Phone
Fax
Email
Web
800-526-3593
877-295-8102
[email protected]
www.gelifesciences.com
GE Healthcare provides tools for drug discovery, biopharmaceutical manufacturing and cellular
technologies so research scientists worldwide can be more productive, effective, and creative.
Our focus is to support the bioprocess researcher from idea to result and be the partner of choice
in cell and protein research.
HORIBA Scientific Booth # 201
3880 Park Avenue
Edison, NJ 08820
Phone
Fax
Email
Web
732-494-8660
732-549-5125
[email protected]
www.horiba.com/scientific
HORIBA Scientific’s SPR Imaging (SPRi) systems utilize SPR to perform multiplexed binding
kinetics measurements of up to 400 proteins, antibodies or DNA in one experiment. Our
Fluorescence line, with the addition of the Photon Technology International (PTI) products,
offers the most extensive array of steady-state, lifetime and microscopy-based fluorometers.
JASCO
Booth # 107
28600 Mary’s Court
Easton, MD 21601
Phone 800-333-5272
Email [email protected]
Web www.jascoinc.com
The new J-1000 Series CD Spectrometers offer unparalleled sensitivity, with reach from the
vacuum UV to the NIR wavelengths. Also, introducing Simultaneous Multi-Probe Spectroscopy
(SMP) which consists of three modes (CD, LD and Absorbance) running concurrently with the
option to run Fluorescence, Anisotropy, ORD, Temperature, Kinetics and more!
49
EXHIBITOR DIRECTORY
JASCO has been the leading manufacturer of CD, VCD and other chiro-optical instrumentation
since the 1960’s. With offices and dealers worldwide we are able to provide the research
community with quality products and experienced support for any application.
Kinnakeet Biotechnology
2336 Colony Crossing Pl
Midlothian, VA 23112
Phone
Fax
Email
Web
Booth # 206
866-522-5338
804-506-4414
[email protected]
www.kinnakeet.com
Since 2003 Kinnakeet Biotechnology has specialized in recombinant viral construction,
amplification, protein expression and purification using the baculovirus protein expression
system. Outstanding customer service and extremely competitive pricing for both small and
large scale production (from 1L to >100L) have assured that >90% of our business comes from
repeat customers.
Malvern Instruments
117 Flanders Road
Westborough, MA 01581
Phone
Fax
Email
Web
Booth # 207
508-768-6400
508-768-6403
[email protected]
www.Malvern.com
Malvern supports better characterization/control of proteins/macromolecules. Charge, size,
mass, molecular weight, polydispersity are critical parameters, measured using: Zetasizer,
dynamic/static light scattering for size, molecular weight, protein charge; NanoSight
Nanoparticle Tracking Analysis, particle-by-particle characterization; Viscotek SEC, molecular
weight/structure; Sysmex FPIA-3000, size/shape analysis of aggregates/subvisible particles;
Viscosizer 200, particle size, concentration, formulation viscosity.
Molecular Dimensions
849 Sunshine Lane
Altamonte Springs, FL 32714
Phone
Fax
Email
Web
Booth # 211
407-886-6901
321-972-8896
[email protected]
www.moleculardimensions.com
Molecular Dimensions founded in 1998 is a world leading supplier of screens, reagents and
instrumentation for protein structure determination by X-ray crystallography.
We have over 100 intelligent solutions for…
Protein Expression, crystal growth screening, custom screens and reagents, crystal growth plates,
cryocrystallography, crystal growth storage, analytical instrumentation, working with crystallographers
for crystallographers.
50
EXHIBITOR DIRECTORY
NACALAI USA, INC.
Booth #102
10225 Barnes Canyon Road, Suite A 103
San Diego, CA 92121
Phone
Fax
Email
Web
858-404-0403
858-404-0408
[email protected]
www.nacalaiusa.com
Nacalai USA, Inc. exhibits the COSMOCORE (Core-Shell particle) and COSMOSIL HPLC
columns, which include analytical and preparative reversed-phase columns, HILIC columns, as
well as unique stationary phases such as the Cholester (cholesteryl group bonded) and pi-NAP
(naphtylethyl group bonded). We also provide high performance magnetic nanoparticles for
chemical biology.
NanoTemper Technologies, Inc.
395 Oyster Point Blvd., Suite 135
South San Francisco, CA 94080
Phone
Fax
Email
Web
Booth # 200
650-763-1658
650-350-4390
[email protected]
www.nanotemper-technologies.com
NanoTemper Technologies develops, produces and markets innovative, high quality
instruments for biomedical research. The products are based on NanoTemper's unique and
proprietary technology, Microscale Thermophoresis (MST), used for the analysis of
biomolecular interactions. NanoTemper offers the Monolith NT series instruments for MST
measurements with fluorescent label and label-free.
PeproTech, Inc.
Booth # 220
5 Crescent Avenue
Rocky Hill, NJ 08553-0275
Phone
Fax
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Web
800-436-9910
609-497-0321
[email protected]
www.Peprotech.com
Over the past 25 years, PeproTech has grown into a global enterprise manufacturing an
extensive line of Recombinant Human, Murine and Rat Proteins, Animal-Free Recombinant
Proteins, Monoclonal Antibodies, Affinity Purified Polyclonal Antibodies, Affinity Purified
Biotinylated Polyclonal Antibodies, ELISA Development Kits, Cytokine Packages and Cell
Culture Media Products.
Pressure BioSciences, Inc.
14 Norfolk Avenue
South Easton, MA 02375
Booth # 104
Phone 508-230-1828
51
EXHIBITOR DIRECTORY
Fax
508-230-1829
Web www.pressurebiosciences.com
PBI develops and sells instrument systems for proteomic, genomic, and lipidomic sample
preparation based on our pressure cycling technology (PCT) platform. PBI is also a distributor of
Constant Systems. (CS) cell disruption equipment for the extraction of proteins and other
biomolecules from organisms such as yeast and bacteria. Two instrument systems will be
showcased (1) PBI’s Barocycler HUB440, capable of reaching 58,000 psi (ideal for protein
extraction, accelerated enzymatic digestion, and protein structure studies), and the CS MultiShot Cell Disrupter, designed for processing samples between 1-80 mL.
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105
Phone
Fax
Email
Web
Booth # 106
901-595-2750
901-595-5376
[email protected]
stjude.org/postdoc
St Jude Children’s Research Hospital is a non-profit biomedical research institution in
Memphis, TN, where 190 basic science and clinical researchers investigate the molecular basis
of both normal cellular and diseased processes. Visit our booth to meet with dedicated
postdoctoral recruiters and to discuss and apply for postdoctoral fellowship positions.
TA Instruments
159 Lukens Drive
New Castle, DE 19720
Phone
Fax
Email
Web
Booth # 208
302-427-4000
302-427-4001
[email protected]
www.tainstruments.com
TA Instruments is the world leader in protein characterization by microcalorimetry. Our Nano
ITC Standard and Low Volume and our Nano DSC with Autosampler are powerful tools for
measuring small-molecule-protein interactions, protein-protein interactions, drug-target
binding, and biomolecular structure and stability. Visit to learn more about the latest
measurement technology.
The Protein Society
Booth # 213
BWTech South Campus
1450 S. Rolling Rd, Ste 3.007
Baltimore, MD 21227
Phone
Fax
Email
Web
443-543-5450
443-543-5453
[email protected]
www.proteinsociety.org
52
EXHIBITOR DIRECTORY
TTP Labtech
Booth # 101
Melbourn Science Park, Melbourn
Royston, Herts, SG8 6EE
Phone +44 1763 262626
Web www.ttplabtech.com
TTP Labtech’s dragonfly screen optimiser is a liquid handler for simple, fast and accurate
protein crystallisation screen optimisation. dragonfly provides the freedom to use any liquid
type, regardless of viscosity, due to its positive displacement, non-contact dispensing. Each
disposable can dispense any volume, from 0.5 µL upwards, into any well.
WILEY
350 Main Street
Malden, MA 02148
Phone
Fax
Email
Web
Booth # 202
781-388-8200
781-338-8212
[email protected]
www.Wiley.com
Wiley is the leading society publisher. Our scientific, technical, medical and scholarly business
publishes on behalf of more societies and membership associations than anybody else, and
offers libraries and individuals 1250 online journals, thousands of books and e-books, reviews,
reference works, databases, and more. For more information, visit www.wiley.com, or our
online resource: onlinelibrary.wiley.com.
53
EXHIBITOR WORKSHOPS
Workshop #1 - GE Healthcare
Sunday, July 27th | Noon - 1 PM | Gaslamp A
NEW Amersham™ 600 Imager Series (CCD-based Camera Systems)
Liz Pita, GE Healthcare, Piscataway, New Jersey, US
Join us to learn more about the newest generation of imaging systems! The Amersham™ 600
series builds upon the robust legacy of ImageQuant™ LAS4000 series that provided high
quality, publication-ready images and reliable research data. Amersham 600 portfolio includes
four systems for routine research applications in pharmaceutical, biotechnology and academic
research including Chemiluminescent and Fluorescent Western blotting, Gel documentation,
DNA and RNA analysis, 2D gels and Colorimetric detection for quantitative and QC analysis.
The new platform complements our Western blotting detection reagents, such as our legacy
Amersham™ ECL as well as Amersham ECL Prime, ECL Select and ECL Plex to provide a
complete Western blotting solution. Please join us to learn more about how our new CCD-based
camera systems can help you in your research!
Workshop #2 – NanoTemper Technologies
Monday, July 28th | Noon - 1 PM | Gaslamp A
Some Like it Hot: Determination of Biomolecular Interactions Using
MicroScale Thermophoresis
Christine Crosby, Ana Lazic, NanoTemper Technologies, S. San Francisco, California, US
This workshop will give an overview of MicroScale Thermophoresis (MST), a technology for
used for the analysis of biomolecule interactions. MST is the directed movement of molecules in
temperature gradients. This thermophoretic movement is determined by the entropy of the
hydration shell around the molecules. Almost all interactions and also any biochemical process
relating to a change in size, charge and conformation of molecules alters this hydration shell and
is thus detectable by MST. In this workshop we will describe the technical details and the
benefits of the MicroScale Thermophoresis technology platform. We will show examples for
interaction measurements ranging from protein – ribosome, protein – protein, small molecule –
receptor down to protein – ion binding studies to experiments where the interactions between
receptors incorporated in vesicles and soluble proteins are analyzed. During the workshop
measurement of interactions will be demonstrated on NanoTemper Technologies’ NT.115 and
NT.LabelFree.
Workshop #3 - GE Healthcare
Tuesday, July 29th | Noon - 1 PM | Gaslamp A
Multimodal Chromatography: A New Generation of High Selectivity Media
for Challenging Purifications
Felix Solamo, GE Healthcare Life Sciences, Piscataway, New Jersey, US
Multimodal chromatography is a powerful tool for difficult separation challenges, including
MAb-aggregate removal. New multimodal resins were developed to address this challenge. The
performance of a multimodal anion exchanger in polishing is shown for two MAb runs in
bind/elute and flow through modes. A multimodal cation exchanger for Mab purification model
was used to choose conditions for the desired purification.
54
INVITED SPEAKER ABSTRACTS
Symposium #1 – Translation & Folding
SYMP 01-21
Policing Secretion: How Cells Enforce Protein Quality Control Within The Endoplasmic Reticulum
Liz Miller, Columbia University, New York, New York, US
The mechanistic basis for quality control surveillance within the secretory pathway remains poorly
understood. We have used a systematic approach to discover proteins that influence the biogenesis of a
model misfolded protein, the yeast ABC transporter, Yor1. Broadly analogous to human CFTR, mutations
in which cause cystic fibrosis, Yor1 acts at the plasma membrane as a drug pump to confer resistance to
oligomycin. Misfolding mutations cause ER retention and proteasomal degradation, leading to oligomycin
sensitivity. This phenotype affords a rapid and robust screen for mutations that enhance or suppress the
ability of cells to tolerate increasing concentrations of drug. Using a high throughput genetic screen, we
measured the effect of each non-essential yeast gene on growth conferred by Yor1-ΛF, equivalent to the
predominant disease-causing allele in CFTR. We discovered novel regulators of membrane protein
biogenesis, including a pathway that seems to detect protein misfolding at the earliest stages of protein
synthesis. We propose misfolding events can generate feedback to the ribosome that halts or slows
translation, serving to either promote folding or prevent aberrant proteins from entering the secretory
pathway. We also discovered an unexpected role for an ER export receptor, Erv14, which seems to
increase the affinity of cargo proteins for the vesicle coat proteins, thereby enhancing ER egress. Genetic
and physical interactions between the various regulators we identified suggest that these events are
coordinated
to
promote
efficient
protein
synthesis,
folding
and
forward
traffic.
SYMP 01-22
Continuous Tracking Of Protein Folding At Microsecond Resolution By A Line Confocal Detection
Of Single Molecule Fluorescence
Satoshi Takahashi, Tohoku University, Japan
Single molecule fluorescence spectroscopy (SMFS) is expected to reveal detailed dynamics involved in
protein folding; however, the time resolution of previous methods of SMFS is typically limited to a few
milliseconds. We recently developed a line-confocal microscope combined with fast sample flow (Oikawa
et al., Sci. Rep. 3, 2151 (2013)), and achieved the time resolution of 20 microsecond in obtaining time
series of FRET efficiency from single molecules. Using the system, we investigated the B domain of protein
A (BdpA), a three-helix bundle, doubly labeled with donor and acceptor fluorophores. We assigned traces
having high and low FRET efficiencies to the native and unfolded states, respectively, confirming the twostate transition of BdpA. The traces assigned to the unfolded state showed a significant fluctuation
occurring in the submillisecond time region. The traces assigned to the native state showed the gradual
shift in the FRET efficiency as the changes in the denaturant concentration. Similar data were obtained for
BdpA labeled at different sites, suggesting the melting of the native state structure. We are currently
improving the system to achieve the better time resolution and the longer observation time. The line
confocal detection of SMFS will become a powerful tool to understand the mechanism of protein folding
and other dynamics as an experimental counterpart of molecular dynamics calculations.
55
SYMP 01-23
Intrinsically Disordered Proteins: Kinetics And Mechanism
Jane Clarke, University of Cambridge, Cambridge, UK (Chair)
Many intrinsically disordered proteins function by folding upon binding to a target protein. It is often said
that IDPs provide high specificity with low affinity, but kinetic analysis of a number of systems suggests
that this is not universally correct. Why then are disordered proteins so ubiquitous? Is disorder in the IDP
important for the function? I will discuss some of our recent kinetic and mechanistic studies of a number
of IDPs that fold upon binding.
Symposium #2 – Bacterial Interactions
SYMP 02-40
The Gram-Negative Cell Envelope as Seen by Protein Antibiotics
Colin Kleanthous, University of Oxford, Oxford, UK
Protein antibiotics are species-specific antimicrobials deployed by Gram-negative bacteria to kill their
neighbours during competition for resources. They are implicated in the stable co-existence of bacteria
within microbiomes and are known to give pathogenic organisms an advantage against commensal
competitors during gut inflammation. My laboratory is exploring the import mechanism of nuclease
colicins, protein antibiotics specific for the model organism Escherichia coli that target its genome,
ribosome or tRNAs. Our aim is to understand how these folded, 60-kDa proteins traverse the entirety of
the Gram-negative cell envelope to deliver their toxic payload. My talk will focus on recent work where we
have been using colicins as probes of fundamental processes in the outer membrane, including
demonstrating how these toxins use an intrinsically disordered domain to pass an epitope signal to the
periplasm through the porin OmpF.
SYMP 02-41
Bacterial Proteins That Modulate Host Membrane Transport Pathways
Craig Roy, Yale School of Medicine, New Haven, Connecticut, US
Bacteria that replicate inside eukaryotic cells typically produce effector proteins having biochemical
activities that manipulate host functions. Many of these bacterial effector proteins are delivered into the
cytosol of host cells and modulate processes important for creating a vacuole that supports intracellular
replication. The biochemical functions of effector proteins from the intracellular pathogens Legionella
pneumophila and Coxiella burnetii that play specific roles in controlling host membrane transport will be
described. These studies reveal new enzymatic activities and protein structures that demonstrate these
pathogens encodes novel effector proteins that can manipulate evolutionarily conserved host proteins
that control membrane transport processes, which provide insight into how bacterial pathogens are able
to construct a unique vacuole inside host cells.
SYMP 02-42
Protein Interactions Regulating Self/Non-Self Recognition in Bacterial Contactdependent Growth
Inhibition
David Low, University of California, Santa Barbara, California, US (Chair)
Bacteria that express contact-dependent growth inhibition systems (CDI) outcompete siblings that lack
immunity, suggesting that these systems mediate intercellular competition. Notably, CDI systems appear
to be restricted to members of the same or closely related species. Analysis of the CDI system from E. coli
56
EC93, which interacts with β-barrel assembly protein BamA, revealed the molecular basis of this
speciesrestriction. The predicted membrane topology of BamA indicates that three of its extracellular
loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity.
Our data indicate that BamA loops 6 and 7 form the CdiAEC93- binding epitope and their variation
between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically
between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAE.coli and
CdiAEC93 play a role in self/non-self discrimination. Self/non-self discrimination further effected once
toxins are injected into neighboring bacterial cells via specific immunity proteins which specifically bind to
their cognate toxins. Structural analysis of toxin-immunity complexes reveals similar protein folds for two
nuclease toxins with little homology between them. Notably, the cognate immunity proteins bind to
different toxin regions to block activity. The self/non-self recognition orchestrated by CDI systems likely
plays an important role in the biology of bacterial communities.
Symposium #3 – Frontier High-Throughput Techniques
SYMP 03-01
Probing Meiotic Specializations to Genome Decoding by Ribosome Profiling
Gloria Brar, University of California, Berkeley, California, US
Meiosis is a complex and well-conserved program of cellular differentiation. We performed genome-wide
measurements of mRNA abundances and new translation using ribosome profiling through yeast meiosis
to better understand the molecular basis for the full cellular restructuring that accompanies meiotic
chromosome segregation. This quantitative and global view of translation through a developmental
process revealed great complexity to the protein complement of these differentiating cells, both in the
number and the structure of expressed genes. Nearly every gene in the yeast genome was translated in
meiotic cells in a strongly stage-specific manner, including disparate and regulated induction of
conserved stress pathways. Translational regulation contributed broadly to this temporal control of
protein synthesis timing through several mechanisms, including the use of competitive upstream Open
Reading Frames (uORFs) on regulated 5’ leader sequences. Additionally, meiotic translation of thousands
of novel short ORFs was observed, expanding our view of what constitutes a coding region even in the
most well annotated eukaryotic genome.
SYMP 03-02
Visualizing Transcription Pausing and Backtracking Genome-Wide at Nucleotide Resolution
L. Stirling Churchman, Harvard Medical School, Boston, Massachusetts, US
It is now clear that transcription is heavily regulated after RNA polymerase has escaped from promoter
regions, mediated largely through transcriptional pausing. These events influence the abundance,
covalent composition and cellular fate of the resulting transcript. Dissecting the pathways and genomic
elements that control transcriptional pausing requires strategies for following in vivo transcription
elongation at similar precision as afforded by in vitro transcriptional assays. Native elongating transcript
sequencing - NET-seq, accomplishes this goal by exploiting the extraordinary stability of the DNA-RNARNA polymerase ternary complex to capture nascent transcripts directly from live cells without
crosslinking. The identity and abundance of the 3’ end of purified transcripts are revealed by deep
sequencing thus providing a quantitative measure of RNAP density with single nucleotide precision.
Application of NET-seq in Saccharomyces cerevisiae reveals pervasive polymerase pausing and
backtracking throughout the body of transcripts. While similar pause frequencies have been observed on
bare DNA, an understanding of their molecular origin has been constrained by the handful of DNA
57
templates that have been studied in vitro. Analysis of pause sites across the yeast genome uncovers key
molecular interactions within the DNA-RNA-RNAP ternary complex that dictate much of the observed
pausing and backtracking dynamics. These interactions and their energetics are at the core of regulatory
processes that use transcriptional pausing to control gene expression.
SYMP 03-03
Structural Analysis of Proteins in Native Environments, Fact or Fiction?
Juri Rappsilber, University of Edinburgh, Scotland, UK
No abstract provided.
SYMP 03-04
A Mass-Spectrometry-Based Map of Universally-Shared Animal Protein Complexes
Ed Marcotte,University of Texas - Austin, Austin, Texas, US (Chair)
An important aspect of a protein’s function is its assembly with other proteins into higher molecular
weight complexes that are typically the active species in the cell. Knowledge of protein complexes often
reveals proteins’ functions, especially when some members of the complex are better characterized than
others. This problem is critically important for the core set of proteins shared by every animal cell, which
form the basic machinery common to every human cell and the cells of many major model organisms;
more than 1/3 of these key proteins are still largely uncharacterized. More generally, maps of protein
complexes provide the mechanistic foundations for understanding diverse human traits and diseases. One
approach that is proving remarkably powerful is based on systematically mapping protein complexes by
native biochemical fractionation and high-throughput mass spectrometry. In this paradigm, protein
complexes are computationally inferred from the separation behavior of proteins across many,
independent biochemical fractionations. We have launched a major effort to apply this strategy to define
the set of major, stable protein complexes shared across animal cells. In initial studies, we separated
cultured human cell extracts into >2,000 biochemical fractions, subsequently analyzed by tandem mass
spectrometry (nearly 9,000 hours of instrument time), thereby enriching and systematically identifying 622
putative native soluble protein complexes and documenting ~14,000 protein interactions. We have now
extended these studies to samples from 7 animal lineages, analyzing >70 biochemical fractionations
comprising >7,000 distinct biochemical fractions, in all capturing the biochemical separation behavior of
~12,000 animal proteins. I’ll describe our progress analyzing these data and our efforts to define the core
set of stable protein complexes conserved across metazoa, as well to measure the evolutionary
conservation, divergence, and rewiring of protein complexes across full animal proteomes.
Symposium #4 – Proteins in Altered States
SYMP 04-05
Protein Aggregation Done Right: The Biogenesis of Functional Amyloids
Matthew Chapman, University of Michigan, Ann Arbor, Michigan, US
Organisms that span nearly every facet of cellular life produce functional amyloids. Microbial functional
amyloids fulfill essential physiological roles for the bug, while also providing a sophisticated suite of
genetic and biochemical tools for understanding how cells coordinate and control amyloid formation.
Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation
and host colonization. Such controlled amyloidogenesis requires that the fiber subunit, CsgA, be
chaperoned prior to secretion to avoid premature oligomerization and associated toxicity. We found that
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CsgC is a highly potent inhibitor of CsgA amyloid formation. The loss of CsgC resulted in the
accumulation of toxic intracellular CsgA amyloid in vivo. Furthermore, CsgC inhibited CsgA amyloid
formation at substoichiometric concentrations by preventing β-sheet-rich oligomerization in vitro. CsgC
also displayed amyloid inhibitory activity against related bacterial client proteins such as CsgA-derived
peptides and sequence homologues. Interestingly, CsgC inhibited amyloid assembly of α-synuclein, but
not Aβ42. We identified a common Q-X-G-X1/2-N-X5-Q motif in CsgC client proteins that is not found
Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid
inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.
SYMP 04-06
Structural Malleability of Intrinsically Disordered Proteins Underlying Alternative Functional States
Peter Tompa, Vrije Universiteit Brussel, Brussels, Belgium
Intrinsically disordered proteins (IDPs) and complex multidomain proteins are characterized by a dynamic
ensemble of conformations that cannot be unequivocally described by traditional static terms of structural
biology (1). Their structural ensemble is dynamic and malleable, enabling them to adapt to a wide variety
of regulatory signals. The quantitative description of structural ensembles has just started (2) and here we
will show that it holds the promise to elucidate complex protein regulatory phenomena (3), such as
moonlighting (4) and allostery (5) in the “supertertiary” structure (6) of proteins. 1) Tompa, P. (2011)
Unstructural biology coming of age. Curr. Opin. Struct. Biol. 21: 419. 2) Varadi, M. et al. (2014) pE-DB, a
database of protein structural ensembles. Nucl. Acids. Res. Nucleic Acids Res. 42: D326. 3) Tompa, P.,
Varadi, M. (2014) Predicting the predictive power of IDP ensembles. Structure 22: 177. 4) Tompa, P., Szász,
Cs. and Buday, L. (2005) Structural disorder throws new light on moonlighting. Trends Biochem. Sci. 30,
484-489 5) Tompa, P. (2014) Multisteric regulation by structural disorder in modular signaling proteins: an
extension of the concept of allostery. Chem. Rev. (Epub ehaed of print) 6) Tompa, P. (2012) On the
supertertiary structure of proteins. Nature Chem. Biol. 18, 597.
SYMP 04-07
Amyloid Assemblies and Their Interactions With Cellular Components
Helen Saibil, Birkbeck College, London, UK
Protein misfolding into amyloid is closely associated with progressive, fatal and as yet incurable
neurodegenerative diseases. We are beginning to understand some of the interaction partners in the
protein quality control systems whose failure leads to aggregation and toxicity, but a general principle is
lacking for the mechanisms and targets of toxicity. We have been using 3D electron microscopy to study
these processes in vitro and in cells. In vitro model of membrane damage: Misfolded oligomers and ends
of fragmented amyloid fibrils have been implicated in damage to cellular membranes. Cryo-electron
tomography of liposomes being attacked by short fibrils of beta-2 microglobulin reveals that binding of
fibril ends to the liposome surface results in distortion and breakage of the membranes, by creating
pointed extrusions and removal of the outer leaflet of the lipid bilayer (Milanesi et al, PNAS 109, 20455,
2012). Structure of toxic Abeta oligomers: Abeta protofibrils, precursors to mature amyloid fibrils, are
correlated with inhibition of synaptic long term potentiation via binding to cellular PrP (Nicoll et al, Nature
Comms 4:2416, 2013). We have used single particle EM and tomography to show that these assemblies
have an unexpected distinctive, flexible helical nanotube structure. Effects of chaperone-disaggregase
systems on yeast prions: Deposition of amyloid aggregates in cells is thought to result from the balance
between protein misfolding/aggregation and the actions of molecular chaperones and disaggregases in
preventing or reversing these processes. In order to examine this balance in vivo, we are studying the
three-dimensional arrangement of model prion aggregates in yeast cells. [PSI+] and [RNQ+] prions are
deposited as clusters of fibrils in distinct, but not membrane-bound, assemblies in the yeast cytoplasm.
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The polymerization of amyloid forming regions and fibril breakdown by disaggregases also results in
transmission of the prion phenotype to daughter cells and mating partners. Using electron tomography of
yeast cell sections containing prions, prepared by high pressure freezing and freeze-substitution or cryo
sectioning, we observe changes in the assembly state of the fibril clusters when levels of the Hsp70
system chaperones or the disaggregase Hsp104 are altered.
SYMP 04-08
Dynamic Complexes, Folding And Phase Separation Of Disordered Proteins In Biological Regulation
Julie Forman-Kay, The Hospital for Sick Children, Toronto, Ontario, Canada (Chair)
Intrinsically disordered proteins (IDPs) and regions play important biological roles, including in translation
and RNA processing. They lack stable structure and can adopt different structural states. Many IDPs are
monomeric or engage in discrete interactions, folding upon binding or retaining significant disorder in the
bound state. The disordered 4E-BP2 interacts tightly with eIF4E to suppress cap-dependent translation
initiation. Binding involves the canonical YXXXXLΦ motif, which undergoes a disorder-to-helix transition,
as well as a secondary site, leading to a dynamic bipartite complex enabling kinase accessibility.
Phosphorylation induces folding of part of 4E-BP2, sequestering the YXXXXLΦ motif and reducing eIF4E
binding. Phosphorylation-induced folding of 4E-BPs exemplifies a new paradigm for IDP-mediated
regulation. Other IDPs are involved in large-scale association having different degrees of order, from more
defined fibers to disordered liquid states. These latter can provide the matrix for cellular membrane-less
organelles. The Ddx4 RNA DEAD-box helicase is involved in one such organelle, germ granules,
functioning in RNA processing in spermatogenesis. When expressed in cells, the protein forms micronsized organelles. In vitro, the disordered N-terminal 250 residues phase separate to form droplets with
similar properties. Perturbations of Ddx4 Phe or Arg residues disrupt phase separation, suggesting a role
for multi-valent cation-pi interactions. Ongoing biophysical studies of Ddx4 will illuminate the
mechanisms of the biogenesis and disassembly of membrane-less organelles. Studies of the variety of IDP
accessible conformational states and their regulated transitions will be critical for understanding
translation and RNA processing, as well as many other cellular functions.
Symposium #5 – Protein Evolution
SYMP 05-24
Evolution in a Test Tube Yields De Novo Enzymes With Unusual Structure and Dynamics
Burckhard Seelig, University of Minnesota, St. Paul, Minnesota, US
Billions of years of natural evolution yielded enzymes that catalyze a wide range of chemical reactions
facilitated by their intricate three-dimensional structures. Despite the rapidly increasing wealth of
information on protein structures we currently have only a limited understanding of how new protein
folds emerge in nature. At the same time, the engineering of entirely new protein scaffolds that are able
to carry out chemical catalysis remains a major challenge in enzyme design. We developed a general
strategy to create artificial enzymes by harnessing the functional diversity of very large libraries of
randomized proteins. We isolated enzymes that catalyze a ligation reaction for which no natural enzymes
are known. The starting library of 4 trillion mutants was based on a non-catalytic zinc finger scaffold. To
our surprise, the new enzymes isolated by in vitro selection and evolution had entirely lost the starting
scaffold and adopted a new fold instead. Different from classic natural proteins, the enzyme lacks
secondary structural motifs and shows high conformational dynamics. Contrary to common belief, the
unique properties of this novel fold demonstrate that a small protein structure with suitable flexibility is
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sufficient to carry out enzymatic function. This example emphasizes the power of an evolutionary
approach, which can yield useful novel enzymes that nature has never seen before.
SYMP 05-25
Sequence-Function-Fitness Landscapes Viewed by Massively Parallel Sequencing Approaches
Dan Bolon, University of Massachusetts Medical School, Worcester, Massachusetts, US
Current sequencing technology enables experimental quantification of mutational landscapes that
describe the functional or fitness effects of all possible point mutations in a gene. These mutational
landscapes provide insights into biochemical and biophysical mechanisms that govern protein evolution.
This presentation will include recent studies of fitness landscapes in yeast, mammalian cells in culture and
influenza virus.
SYMP 05-26
Evolution of Novel Components of the Bacterial Flagellar Motor
Morgan Beeby, Imperial College London, London, UK
Bacteria propel themselves in beneficial directions using cell wall-embedded rotary motors that spin
helical propellers called flagella. Studies of the Escherichia coli motor have revealed a large nanomachine
composed of hundreds of proteins — from approximately twenty protein families — that harness protonmotive force to produce torque. Using electron cryo-tomography to perform 3D in situ imaging we
recently discovered that many bacteria have motors considerably more complex than the ‘normal’ core
motor found in E. coli. Why have some bacteria recruited additional proteins to a core that is already fully
functional? I will describe phylogenetic and structural work to understand this question. We are currently
focusing on a family of large novel structural components found in a variety of proteobacteria including
the Vibrio, Campylobacter, and Helicobacter genera. By combining bacterial genetics, phylogenetics and
electron cryo-tomography we have identified the proteins that form these large structures. Combining
these results we have developed a model for the recruitment of these additional proteins, and have been
able to speculate on the effect of these proteins on motor mechanics, in turn highlighting possible
evolutionary driving forces behind protein recruitment.
SYMP 05-27
The Remarkable Pliability and Promiscuity of Specialized Metabolism
Joseph P. Noel, Salk Institute for Biological Studies/HHMI, La Jolla, California, US (Chair)
Symposium #6 – Proteins in Disease & Therapeutics
Sponsored by Bristol-Myers Squibb
SYMP 06-43
Structural Basis of Broad Neutralization of Viral Pathogens
Ian Wilson, The Scripps Research Institute, La Jolla, California, US
Influenza, Hepatitis C, and HIV-1 continue to constitute significant threats to global health. We have
structurally and functionally characterized their viral antigens and interaction with broadly neutralizing
antibodies (bnAbs). A number of antibodies have recently been identified that are extremely potent and
neutralize across multiple subtypes and types of these viruses through binding to functionally conserved
sites, such as the receptor binding site or the fusion domain. For HIV-1, novel epitopes involve glycans.
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The glycan-dependent Abs have unique features that enable them to penetrate the glycan shield on the
viral Env protein and bind extensive and complex epitopes that consist of sugars and underlying protein
segments on gp120 on HIV-1 Env. This structural information is now being used to aid in vaccine design
for HIV-1, HCV and for a more universal flu vaccine. IAW is supported by NIH grants AI100663, AI082362,
AI84817, AI099275 and GM094586 and the Crucell Vaccine Institute.
SYMP 06-44
The HIV-1 Viral Spike: Conformational Machine for Entry and Evasion
Peter Kwong, NAIAD - NIH, Bethesda, Maryland, US
The HIV-1 envelope (Env) glycoprotein spike, which comprises three gp120 and three gp41 subunits, is a
type 1 fusion machine, which uses the energy stored in its metastable fold to facilitate virus-host entry by
fusing viral and cellular membranes. The prefusion spike exists in at least two conformations: a
neutralization-resistant ground state and a CD4 receptor-bound intermediate state, while the postfusion
spike consists of only the gp41 subunit, with the gp120 subunit having been shed from the virion surface
during the fusion process. Over the past 20 years, substantial structural information has been obtained on
both gp120 and gp41, in pre- and postfusion conformations. However the prefusion ground state has
resisted atomic-level analysis. Here we used neutralizing antibodies PGT122 and 35O22 to capture the
viral spike in its ground state. Crystals were obtained of these two antibodies in complex with a soluble
Env trimer construct (BG505.SOSIP. 664), and the structure determined to 3.5 Å resolution. Prefusion gp41
folds around extended N- and C-terminal beta-strands of gp120. Comparison of ground state and CD4bound conformations of gp120 reveal the structural rearrangements induced by receptor. In addition to a
description of the trimeric ground-state structure of the HIV-1 Env ectodomain, implications for HIV-1
vaccine design will be discussed.
SYMP 06-45
Jamie Rossjohn, Monash University, Clayton, Victoria, Australia
The T cell receptor complex, expressed on the surface of T-cells, comprises the antigen-specific
heterodimeric αβ T-cell receptor (TCR) that is associated with the CD3 complex. Ligation of the TCR by an
Antigen (Ag) presenting molecule initiates T-cell signalling. While is conventionally considered that TCRs
interacts with peptides bound to the Major Histocompatibility Complex (MHC), TCRs can also bind lipidbased Ags bound by CD1 family members. Moreover, we have recently established how T-cells, namely
Mucosal-associated invariant T-cells, can recognise microbial-based vitamin B metabolites when bound to
the MHC-I-like molecule, MR1 (refs 1,2) . I shall discuss the structural basis of vitamin B metabolite
presentation and recognition of this MAIT TCR-MR1 system.
SYMP 06-46
Pamela Bjorkman, California Institute of Technology/HHMI, Pasadena, California, US
Over 30 years after the emergence of HIV-1, there is no effective vaccine, and AIDS remains an important
threat to global public health. Following infection by HIV-1, the host immune response is unable to clear
the virus due to a variety of factors, including rapid viral mutation and the establishment of latent
reservoirs. The only target of neutralizing antibodies is the trimeric envelope (Env) spike complex, but HIV1 can usually evade anti-spike antibodies due to rapid mutation of its two spike glycoproteins, gp120 and
gp41, and structural features that allow the spike to hide conserved epitopes. Because a completely
protective vaccine against HIV has not been found, possible prevention/treatment options involving
delivery of broadly neutralizing antibodies (bNAbs) identified in a minority of HIV-infected individuals are
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being considered. bNAbs that target conserved epitopes on the HIV envelope spike can prevent infection
in animal models, delay rebound of HIV after cessation of anti-retroviral drugs, and treat an ongoing
infection. Enhancing the efficacy of bNAbs; in particular, designing bNAbs that retain potency against
escape mutants selected during exposure to bNAbs, would facilitate their use as therapeutics. We
previously used structure-based design to create NIH45-46G54W, a CD4-binding site (CD4bs) antibody
with superior potency and/or breadth compared with other bNAbs. Here we report even more effective
variants of NIH45-46G54W designed using analyses of the NIH45-46/gp120 complex structure and
sequences of antibody-resistant HIV clones. One mutant, 45-46m2, neutralizes 96% of HIV strains in a
cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known
bNAbs, making it the single most broad and potent anti-HIV antibody to date. A detailed description of
its mechanism is presented based on a 45-46m2/gp120 crystal structure. A second mutant, 45-46m7,
designed to thwart resistance from NIH45-46G54W due to mutations in a V5/loop D gp120 consensus
sequence, restores neutralization of HIV consensus sequence mutants, thus effectively targeting a
common route of HIV escape. In combination, almost all HIV isolates are effectively neutralized, reducing
the possible routes for the evolution of fit viral escape mutants.
Symposium #7 – Chemical Biology & Enzymology
SYMP 07-09
Spatially-Resolved Proteomic Mapping of Mitochondria in Living Cells Using an Engineered
Peroxidase Reporter
Alice Ting, Massachusetts Institute of Technology, Cambridge, Massachusetts, US
SYMP 07-10
2014 P ROTEIN SCIENCE BEST PAPER AWARD WINNER TALK 1
Mark Landau, Yale University, New Haven, Connecticut, US
Mark J. Landau, Hitesh Sharma, Karen S. Anderson (2013)
Selective Peptide Inhibitors of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase From
Toxoplasma Gondii Provide Insights Into Domain-Domain Communication and Allosteric
Regulation. Protein Sci. 22:1161-1173.
SYMP 07-11
Structure-Based Screens for Protein De-orphanization
Brian Shoichet1, 2, 1University of Toronto, Toronto, Ontario, Canada, 2University of California-San
Francisco, San Francisco, California, US
For only small fraction of sequenced genes is the function of their encoded proteins known, or even
predicted with confidence. Here we investigate a structure-based approach to predicting enzyme and
receptor activity. Libraries of metabolites or drug-like molecules are screened against the structures of
enzymes or G Protein-Coupled Receptors (GPCRs), respectively, and high-scoring molecules are tested
experimentally as either substrates or agonists/antagonists/allosteric modulators. This approach has
discovered substrates against enzymes of the amidohydrolase and enolase superfamilies. Very recently we
have expanded this work to orphan GPCRs, in collaboration with Bryan Roth’s laboratory. A combined
experimental and computational strategy to deorphanize GPR68 will be discussed, with applications to the
role of this orphan GPCR in vivo, in a mouse model.
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SYMP 07-12
Molecular DNA Devices in Living Systems
Yamuna Krishnan, National Center for Biological Sciences, Bangalore, India
Due to its nanoscale dimensions and ability to self-assemble via specific base pairing, DNA is rapidly
taking on a new aspect where it is finding use as a construction element for architecture on the
nanoscale.1 Structural DNA nanotechnology has yielded architectures of exquisite complexity and
functionality in vitro. However, till 2009, the functionality of such synthetic DNA-based devices in living
organisms remained elusive. Work from my group the last few years has bridged this gap where, we have
chosen architecturally simple, DNA-based molecular devices and shown their functionality in complex
living environments. Using two examples, from our lab, one of a rigid, DNA polyhedron2 and the other a
molecular switch3 that functions as a pH sensor I will illustrate the potential of DNA based molecular
devices as unique tools with which to interrogate living systems.
SYMP 07-13
Activity-based Proteomics - Applications for Enzyme and Inhibitor Discovery
Benjamin Cravatt, III, The Scripps Research Institute, La Jolla, California, US (Chair)
Genome sequencing projects have revealed that eukaryotic and prokaryotic organisms universally possess
a huge number of uncharacterized enzymes. The functional annotation of enzymatic pathways thus
represents a grand challenge for researchers in the genome era. To address this problem, we have
introduced chemical proteomic and metabolomic technologies that globally profile enzyme activities in
complex biological systems. These methods include activity-based protein profiling (ABPP), which utilizes
active site-directed chemical probes to determine the functional state of large numbers of enzymes in
native proteomes. In this lecture, I will describe the application of ABPP and complementary proteomic
methods to discover and functionally annotate enzyme activities in mammalian physiology and disease. I
will also present competitive ABPP platforms for developing selective inhibitors for poorly characterized
enzymes and discuss ongoing challenges that face researchers interested in assigning protein function
using chemoproteomic methods.
Symposium # 8 – Cellular Structures
SYMP 08-14
New Insights Into Microtubule Mechanics
Manuel Théry1, 2, 3, 1LPCV, Grenoble, France, 2iRTSV, Grenoble, France, 3DSV / CEA, Grenoble, France
Tubulin dimers self-organize into hollow tubes called microtubules. These microtubules support
numerous cellular functions such as intra-cellular transport or mitotic spindle assembly. Therefore the
architecture of the microtubule network is essential in the regulation of cell physiology. Unfortunately our
understanding of microtubule mechanical properties is surprisingly limited. Indeed the lack of
appropriated experimental approaches has hampered our capacity to investigate these properties. The
thermal fluctuations of microtubules in cell-free extracts have shown that they were extremely rigid, with a
persistent length a thousand times larger than cell size. However they appear highly curved in cells which
somehow contradict in vitro measurements. We developed a new microfluidic device to apply controled
constraint on microtubules and observed their bending properties. Thereby we revealed new and
unexpected features of microtubule mechanics.
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SYMP 08-15
LORNE CONFERENCE ON PROTEIN STRUCTURE AND FUNCTION EXCHANGE SPEAKER
Protein Degradation in Bacteria and Mitochondria: When the N-Terminus Signals the End
Kaye Truscott, La Trobe Institute for Molecular Science, Melbourne, Victoria, Australia
SYMP 08-16
A Protein Interaction Network That Directs Human Cytoplasmic Dynein to Microtubule Ends
Thomas Surrey1, 2 , 1London Research Institute, London, United Kingdom, 2Cancer Research UK, London,
United Kingdom
Growing microtubule ends recruit several proteins collectively called +TIPs which confer local functions to
the microtubule cytoskeleton. These +TIPs form dynamic protein interaction networks with competitive
and hierarchical interactions. The rules that determine which of the multiple +TIPs bind to the limited
number of available binding sites in microtubule end regions are only poorly understood. Here we
investigated how the major human minus end directed motor dynein that is also an important +TIP, is
targeted to growing microtubule ends in the presence of competitors. Using a TIRF microscopy-based in
vitro reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin
subunit p150glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP
motif-containing peptides. The human dynein complex is then targeted to growing microtubule ends
through an interaction of the tail domain of dynein with p150glued. Our results show how the
connectivity and hierarchy within dynamic +TIP networks are orchestrated.
SYMP 08-17
Mechanisms of Mitosis and Intracellular Scaling in Xenopus
Rebecca Heald, University of California-Berkeley, Berkeley, California, US (Chair)
The goal of my laboratory is to elucidate the molecular mechanisms of cell division and morphogenesis.
We use cytoplasmic extracts prepared from eggs of the frog Xenopus laevis to reconstitute and study
mitotic chromosome condensation and spindle assembly and function in vitro, applying imaging,
biochemical, and biophysical approaches. To study mechanisms of spindle and organelle size control, we
take advantage of a smaller, related frog, Xenopus tropicalis, to investigate interspecies scaling, and
extracts prepared from fertilized eggs at different stages of embryogenesis to study developmental
scaling. Our research will provide novel insight into how cell/organelle scaling contributes to intracellular
morphogenesis and cell division, processes essential for viability and development, and defective in
human diseases including cancer.
Symposium #9 – Protein Engineering & Synthetic Biology
SYMP 09-28
No title provided
Christopher A. Voigt, Massachusetts Institute of Technology, Cambridge, Massachusetts, US
SYMP 09-29
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Sustaining Life With Proteins Designed De Novo
Michael Hecht, Princeton University, Cambridge, Massachusetts, US
The collection of all protein sequences that ever existed on earth represents a tiny fraction of the
sequence space that is possible. From the enormous diversity of possible sequences, nature has selected
very small collections of 'molecular parts' to sustain life: Only ~4,000 genes in E. coli and ~23,000 in
humans. These considerations may lead to the assumption that natural sequences are somehow special. Is
this true? Or can sequences designed ‘from scratch’ also provide essential functions necessary to sustain
life? To address these questions, we designed and constructed millions of artificial proteins encoded by a
library of synthetic genes. Structural studies show that many of our novel proteins fold into stable 3dimensional structures. Biochemical assays demonstrate that many of them bind biologically relevant
molecules, and genetic studies show that several of these novel proteins function in vivo to provide
functions necessary to sustain the growth of E. coli. These results suggest that (i) the molecular toolkit for
life need not be limited to genes and proteins that already exist in nature; (ii) artificial genomes and
proteomes can be built from non-natural sequences; and (iii) synthetic organisms relying on de novo
designed proteins may be possible.
SYMP 09-30
Metal-Directed Protein Evolution
Akif Tezcan, University of California-San Diego, San Diego, California, US
Metals, especially those of the d-block (i.e., transition metals), play irreplaceable roles in biology,
stabilizing protein structures and enabling biochemical transformations that would be impossible in their
absence. Given their dual, structure- and function-building power, it is natural to ask whether transition
metals can also accelerate/catalyze the evolution of proteins. In this presentation, I will talk about our
group's efforts in exploiting metal coordination chemistry for building and evolving new biological
structures and functions.
SYMP 09-31
De Novo Protein Structures and Assemblies by Design
Dek Woolfson, University of Bristol, Clifton, United Kingdom (Chair)
We have developed a toolkit of de novo peptides (1). These can be used as building blocks for the rapid
construction of new protein-like structures and supramolecular assemblies. This talk will illustrate the
utility of this approach with two examples for making nanoscale peptide-based pores (2) and selfassembled cages (3). Potential applications of the structures and materials achieved span nanoscience,
synthetic biology and biotechnology.
1 .A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology
JM Fletcher et al. ACS Synthetic Biology 6, 240-250 (2012) 2 .A de novo peptide hexamer with a mutable
channel NR Zaccai et al. Nature Chemical Biology 7, 935-941 (2011) 3. Self-assembling cages from coiledcoil peptide modules. JM Fletcher et al. Science 340, 595-599 (2013)
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Symposium #10 – Proteins in Dynamic & Driven Processes
SYMP 10-32
Deciphering Protein Dynamics During Endocytic Budding by Time-Resolved Electron Microscopy
Maria Isabel Geli Fernandez-Penaflor, Molecular Biology Institute of Barcelona, Barcelona, Spain
Endocytic membrane budding from the plasma membrane requires the coordinated recruitment and
function of more than 50 proteins which concentrate cargo, deform the lipid bilayer and effect fission to
generate the primary endocytic vesicle. Molecular models explaining the process are mainly based on the
information derived from the live-cell fluorescence microscopy, which offers a regular resolution of about
200 nm, and the structural and biochemical information of the purified components involved. However,
understanding how endocytic proteins really effect membrane deformation within the cells would require
the dynamic view of the process at nearly atomic resolution. Even though this goal is far from reachable at
the moment, we have made an effort to fill the resolution GAP between the fluorescence microscopy and
the structural biology by developing a methodology that integrates our knowledge derived from the livecell imaging with the statistical processing of parameters describing the shape of endocytic invaginations
and the position of gold particles labelling 18 different endocytic proteins, on ultrathin section of the
yeast S. cerevisiae. This approach has allowed us to describe the dynamics of the endocytic machinery
coupled to the deformation of the lipid bilayer with a resolution down to 7 nm, solving important issues in
the field such as the point of emergence of membrane curvature relative to the recruitment of the
endocytic proteins or the exact function of actin polymerization, the myosin motor activity or the BAR
proteins during vesicle budding.
SYMP 10-33
2014 P ROTEIN SCIENCE BEST PAPER AWARD WINNER TALK 2
Brian Ziemba, University of Colorado, Boulder, Colorado, US
Brian P. Ziemba, Emma J. Murphy, Hanna T. Edlin, David N.M. Jones (2013) A Novel Mechanism of
Ligand Binding and Release in the Odorant Binding Protein 20 From the Malaria Mosquito
Anopheles Gambiae. Protein Sci. 22:11-21.
SYMP 10-34
Spatial Regulation of Molecular Motors
Samara Reck-Petersen, Harvard Medical School, Boston, Massachusetts, US
Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first
step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus
end, but the molecular mechanism underlying this spatial regulation is not understood. In this work we
reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the
mechanism using single-molecule microscopy. We find that two proteins - homologs of Lis1 and Clip170
– are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the
microtubule plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion
though its microtubule-binding domain. Two microtubule-associated proteins, homologs of Clip170 and
EB1, act as processivity factors for Kip2, helping it overcome dynein’s intrinsic minus-end-directed motility.
This reveals how a minimal system of proteins transports a molecular motor to the start of its track.
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SYMP 10-35
The Bacterial Magnesium Channel CorA – Dynamic Ways to Translocate Divalent Cations
Emil F. Pai, University of Toronto, Toronto, Ontario, Canada
The first crystal structure of Thermotoga maritima CorA (TmCorA) determined in the presence of high
concentrations of divalent ions revealed a rotationally symmetric pentamer [Lunin et al., Nature, 440, 833;
Eshagi et al., Science, 313, 354; Payandeh & Pai, EMBO J, 25, 3762, 2006]. Although amino acids crucial in
gating and regulation could be identified [Payandeh et al., JBC, 283, 11721, 2008] the detailed molecular
mechanism of channel opening remains unclear. Initial MD calculations pointed to an iris-like movement
[Chakrabarti et al., Biophys J, 98, 784, 2010] upon channel opening. In the crystal structure of a mutant
CorA, a hydrated Mg2+ ion binds to the periplasmic GMN motif, revealing clues of selectivity. Without
Mg2+, TmCorA displays radial and lateral tilts of protomers that lead to an asymmetric arrangement of
subunits and bending of the central, pore-lining helix. MD simulations support these movements,
including a most unusual bell-like deflection. Mass spectrometric analysis confirms that major proteolytic
cleavage and disulfide formation occur within a region that is selectively exposed by such a motion [Pfoh
et al., PNAS, 109, 18809, 2012]. A recent crystal structure of a pentameric arrangement of the cytosolic
part of TmCorA suggests counterbalancing forces located in the trans-membrane part of the channel
helices and the binding sites of divalent ions between the subunits. Our results indicate a sequential
allosteric model of regulation; intracellular Mg2+ binding locks TmCorA in a symmetric, transportincompetent conformation and loss of intracellular divalent ions causes an asymmetric, potentially influxcompetent conformation.
SYMP 10-36
Engineering Proteins for Visualization and Control of Signaling Networks In Vivo
Klaus Hahn, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, US (Chair)
Cell motility requires the orchestration of many dynamic cellular systems, which can only be fully
understood through quantitation of protein activity in living cells and animals. This talk will describe new
tools to visualize and manipulate signaling in vivo, using Rho family GTPase networks and cell motility as
test beds. Multiplexed imaging of biosensors and computational image analysis will be used to examine
the coordination of GTPases. The role of specific protein activation events regulating coordination will be
probed using engineered allosteric switches and different methods to control the dynamics of protein
activity with light. New technologies will include methods to direct activated kinases to specific targets,
broadly applicable methods to control sequestration of proteins at intracellular membranes with light, and
improvements in biosensor design for applications in animals.
Symposium #11 – Protein Degradation
SYMP 11-18
Ubiquitin-Dependent Regulation of Proliferation and Differentiation
Michael Rape, University of California-Berkeley, Berkeley, California, US
Abstract not provided.
SYMP 11-19
Investigating the Mechanisms of the Proteasome by Cryo-EM
Paula da Fonseca, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
68
In eukaryotes the ubiquitin/proteasome pathway is responsible for the controlled targeting and
degradation of a wide range of proteins, including key cellular regulators such as those controlling cell
cycle progression and apoptosis. The 26S proteasome is a large ATP-dependent protease responsible for
the highly regulated degradation of proteins specifically targeted by ubiquitin. It comprises a proteolytic
20S core associated to 19S regulatory particles, which specifically recognise, unfold and translocate the
substrate proteins into the proteolytic chamber. The 26S proteasome is a well-recognised target for
cancer therapy and its deregulation is associated with neurodegenerative conditions such as Alzheimer’s
and Parkinson’s diseases. However, its functional mechanisms are still significantly elusive. We determined
the structure of the human 26S proteasome by electron cryo-microscopy (cryo-EM) and single particle
analysis where secondary structure elements are clearly identified. By combining our cryo-EM map with
data from x-ray crystallography and structural modelling we revealed the organisation of the 19S
regulatory particle subunits and presented a molecular model for the complete human 26S proteasome.
Through recent developments in the field of structural electron microscopy, triggered by the availability of
improved electron microscopes and direct detectors, it is now possible to achieve resolutions that used to
be attainable only by crystallography or NMR methods. Such developments are now being used to
explore the functional mechanisms of the proteasome.
SYMP 11-20
Application of Small Molecule Probes to the Study of Protease Function
Matthew Bogyo, Stanford University, Stanford, California, US
Proteases are enzymes that primarily function by degrading protein substrates. Since this process is
irreversible, proteases must be carefully regulated within cells and organisms in order to prevent
undesired consequences. Furthermore, proteases often play pathogenic roles in common human diseases
such as cancer, asthma, arthritis and atherosclerosis. Over the past decade, my laboratory has developed a
series of small molecule activity-based probes (ABPs) that specifically bind to the active form of protease
targets through an enzyme catalyzed chemical reaction. These reagents can be used to enrich complex
proteomic samples for monitoring of global patterns of protease activity as well as to directly image
protease activity in live cells and whole animals. They can also be used to monitor the efficacy and
selectivity of small molecule drugs. We are currently applying these probes to study the role of specific
proteases in tumor growth and metastasis in mouse models of cancer as well as during the process of
inflammation in mouse models of atherosclerosis, asthma and pulmonary fibrosis. In addition, we have
applied covalent small molecules to identify important regulators of parasite pathogenesis. Recent
advances in these projects will be presented.
Symposium #12 – Membrane Proteins & Receptors
SYMP 12-37
Mechanism-based Tuning of Cytokine Receptor Signaling
Chris Garcia, Stanford University School of Medicine/HHMI, Stanford, California, US
In my lecture I will address a fundamental question in receptor biology: Does the dimeric geometry of a
ligand-receptor complex influence signaling ? or is receptor dimerization a signaling “on/off” switch ? In
particular, Cytokines are known to dimerize their receptor ectodomains to initiate signaling, but it is
unclear how receptor dimer architecture impacts signaling, or if dimer geometry can be manipulated to
‘tune’ signaling. In order to directly interrogate this question, we have modulated cytokine receptor
signaling by altering the orientation and proximity of a cytokine receptor dimer using diabodies as
69
surrogate ligands. We directly correlate the intact receptor dimer structures, determined
crystallographically, with functional outcome and we find that indeed such systems are ‘tunable’ by
remodeling dimer topology and distance. Furthermore, we show that these extracellular-directed
diabodies can overcome intracellular oncogenic mutations in Jak2. Thus, induced alterations in receptor
dimer geometry can ‘tune’ signaling and extracellular ligands that enforce large receptor inter-subunit
distances can counteract intracellular oncogenic ligand-independent receptor activation.
SYMP 12-38
From Liposomes to Fliposomes: In Vitro Reconstitution of Lipid-Dependent Dual Topology and
Post-Assembly Topological Switching of a Membrane Protein
William Dowhan, University of Texas Houston Medical School, Houston, Texas, US
The mechanism by which membrane proteins exhibit structural and functional duality in the same
membrane or different membranes is unknown. Systematic alteration of membrane lipid composition
uncovered a role for direct lipid-protein interactions in determining initial topogenesis of and dynamic
post-assembly topological changes in protein transmembrane domains (TMDs). Using an in vitro
proteoliposome system in which lipid composition can be systematically controlled before (liposomes)
and after (fliposomes) protein reconstitution, we determined the minimum and sufficient requirements for
a membrane protein to reorganize between topologically distinct states. Increasing or decreasing
phosphatidylethanolamine levels after initial protein reconstitution resulted in re-orientation of the
original mixture of native and inverted topological conformers to favor the native or inverted protein
conformer, respectively. Therefore, reversible “flipping” of TMDs is dependent on direct lipid-protein
interactions independent of molecular chaperone or translocon involvement. Using Förster resonance
energy transfer methods, we determined that the rates of TMD flipping in both directions were on a
second scale and occurred rapidly after proteoliposome lipid composition was changed. These
observations demonstrate a potential thermodynamically driven biological switch for generating dynamic
structural and functional heterogeneity for a protein within cells dependent only on the properties of the
protein and its lipid environment. Funded by NIH grant GM20478 (WD).
SYMP 12-39
Structural and Mechanistic Diversity of ABC Transporters
Douglas Rees, California Institute of Technology/HHMI, Pasadena, California, US
ATP Binding Cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane
proteins responsible for the ATP powered membrane translocation of a wide variety of substrates. The
highly conserved ABC domains defining the superfamily provide the nucleotide-powered engine that
drives transport. In contrast, the transmembrane domains creating the translocation pathway are more
variable, with three distinct folds currently recognized. Structural analyses of the high affinity methionine
MetNI importer and of a bacterial homologue of the mitochondrial Atm1 exporter will be discussed within
the mechanistic framework of the alternating access model. The interconversion of outward and inward
facing conformations of the translocation pathway is coupled to the switching between open and closed
interfaces of the ABC subunits that are associated with distinct nucleotide states. As observed for MetNI,
additional domains may be present that can regulate transport activity. Building on this qualitative
molecular framework for deciphering the transport cycle, an important goal is to develop quantitative
models that detail the kinetic and molecular mechanisms by which ABC transporters utilize the binding
and hydrolysis of ATP to power substrate translocation.
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POSTER ABSTRACTS
Poster Session: Proteins in Disease & Therapeutics
POST 10-50
Coupling the Antimicrobial Action of a Novel Un-natural Cationic Amphiphilic Polyproline Helix
with its Cell Penetrating Ability to Target Intracellular Bacteria
Manish Nepal , Jean Chmielewski, Purdue University, West Lafayette, Indiana, US
The rise and emergence of multidrug resistance bacteria requires advancements in novel anti-microbial
agent design. An additional challenge to development of anti-microbial agent is that bacteria such
as Salmonella, Listeria, and Brucella reproduce inside mammalian cells and are inaccessible to many
antimicrobial drugs (β-lactams and animoglycosides). Herein we present a cationic amphiphilic
polyproline helix (CAPH), Fl-PLPRPR-5, that exhibits antibacterial action against a broad spectrum of
pathogenic Gram positive (Listeria monocytogenes; MIC 8 µM) and Gram negative (Acinetobacter
baumannii; MIC 2 µM) bacteria. Additionally, the agent also displays superior cell penetration with the
macrophage cells; the “safe haven” for some of the intracellular resistant bacterial strains. Preliminary
studies have confirmed the internalization of Fl-PLPRPR-5 in E. coli and its non-membrane lytic mode of
action. Fl-PLPRPR-5 demonstrates minimal hemolysis of human erythrocyte cells and also resists enzymatic
degradation against trypsin. Fl-PLPRPR-5 rescued J774A.
POST 10-51
Rational Design of Thefurin Cleavage Site of an Anti-CD22 Recombinant Immunotoxin Based on
Pseudomonas Exotoxin A
John E. Weldon1, 2, Ira Pastan2
1
Department of Biological Sciences, Towson University, Towson, Maryland, US, 2Laboratory of Molecular
Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, US
Recombinant immunotoxins (RITs) are fusion proteins that combine antibodies with protein toxins in
order to selectively target and kill cells. RITs that join antibodies against tumor-associated antigens with
the bacterial toxin Pseudomonas exotoxin A (PE) are currently undergoing clinical trials for the treatment
of cancers, and have achieved some notable successes. The general utility of RITs, however, is limited by
several factors that include the requirement for a suitable antigen target, the formation of neutralizing
antibodies in patients, and the necessity for potent targeted activity on tumor cells. These and other
limitations are areas of active exploration to improve the efficacy of PE-based RITs. One potential
enhancement strategy involves rational engineering of RITs utilizing our understanding of the PE
intoxication pathway. During intoxication PE is internalized by receptor-mediated endocytosis, traffics
through the endolysosomal system to the Golgi, and undergoes retrograde transport to the endoplasmic
reticulum. PE is subsequently exported into the cytoplasm, where it encounters and ADP-ribosylates
elongation factor 2, halting protein synthesis and leading to cell death. A step in the pathway that may be
amenable to protein engineering is the cleavage of PE by the intracellular protease furin during
endolysosomal trafficking. We suspect that furin cleavage may be a rate-limiting step in the intoxication
pathway of PE, and have targeted the furin cleavage site for engineering in an effort to enhance the
cytotoxicity of PE-based RITs. Our findings suggest that furin cleavage efficiency and RIT cytotoxicity are
not directly correlated, but overall cytotoxicity improvements warrant further investigation.
POST 10-52
Structural and Functional Analysis of Lysosomal Phospholipase A2, a Close Homolog of LecithinCholesterol Acyltransferase
3, 1
2
2
2, 4
4, 1, 5
Alisa Glukhova , Robert J. Kelly , Vania Hinkovska-Galcheva , James A. Shayman , John J. Tesmer
71
POSTER ABSTRACTS
1
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, US, 2Department of Internal
Medicine, University of Michigan, Ann Arbor, Michigan, US, 3Program in Chemical Biology, University of
Michigan, Ann Arbor, Michigan, US, 4Department of Pharmacology, University of Michigan, Ann Arbor,
Michigan, US, 5Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, US
Lysosomal phospholipase A2 (LPLA2, PLA2 group XV) is a recently discovered acyltransferase that
catabolizes glycerophospholipids and breaks down lung surfactants. Inhibition of LPLA2 may be the basis
for the phospholipidosis observed with long-term administration of amiodarone, a treatment for
ventricular arrhythmia. LPLA2 has 50% sequence identity with lecithin-cholesterol acyltransferase (LCAT),
an HDL-associated plasma protein that regulates reverse cholesterol transport. Here we present the 1.83 Å
structure of human apo-LPLA2 and a 2.3 Å structure of LPLA2 treated with an irreversible inhibitor
isopropyl dodec-11-enylfluorophosphonate, which represents an acyl transfer intermediate. LPLA2 is an αhydrolase with additional lid domains responsible for substrate recognition and specificity. LPLA2 also
possesses a membrane binding motif, consisting of a hydrophobic patch surrounded by positively
charged region. The wide opening of the LPLA2 active site faces the membrane for an easy access to
glycerophospholipids and lipophilic alcohols, its preferred substrates. Based on these structures, substrate
modeling, and the position of disease-causing mutations in LCAT, we propose that orientation of the
bound phospholipid in the active site underlies the specificity of LPLA2 for fatty acids in the sn-2 vs. sn-1
position, and that preference for length of the acyl chains is dictated by two hydrophobic grooves leading
away from the catalytic triad of the enzyme. We modelled the structure of LCAT and mapped genetic
mutations leading to either fish eye disease (FED) or familial LCAT deficiency (FLD). We propose that total
loss of enzyme activity in most FLD cases is caused by mutations in residues playing a structural role. On
the other hand, FED mutations mostly cluster on the surface of the enzyme and presumably affect its
activation by HDL particles.
POST 10-53
Structural Basis for Antigen Recognition of a Tumor Specific Therapeutic Antibody
Reza Movahedin, Teresa M. Brooks, Cory L. Brooks
Chemistry , California State University, Fresno, California, US
Antibody mediated immunotherapy of tumors has the potential to revolutionize cancer
treatment.Specificity of monoclonal antibodies (MAbs) allows them to specifically target tumor cells.A
universal feature of cancer cells is aberrant protein glycosylation, results in exposure of tumor specific and
new epitopes that are normally masked that are attractive targets for therapeutic MAbs. MUC1 is a
membrane glycoprotein found in epithelial cells.The with a large extracellular domain called the VNTR
(variable number of tandem repeats) composed of a 20-120 repeating segments of 20 amino acids.The
VNTR region is heavily O-glycosylated in healthy tissues.during neoplastic transformation the VNTR
region of MUC1 exhibits truncated glycosylation, exposing the peptide and truncated carbohydrate .The
aberrantly glycosylated MUC1 is overexpressed in the majority of adenocarcinomas.These features render
MUC1 an ideal target for antibody-mediated immunotherapy. MAb-AR20.5 is a therapeutic antibody
currently undergoing clinical development for treatment of MUC1 positive pancreatic cancer.The antibody
was generated by immunization with tumor-derived MUC1 and exhibits potent antitumor activity. The
antibody binds to a peptide epitope within the VNTR region of MUC1, molecular details of this interaction
are uncharacterized and it is currently unknown what role antigen, glycosylation plays in antibody binding.
To further our understanding of how this antibody interacts with antigen, we have purified and
crystallized the Fab fragments of AR20.5 in complex with a synthetic MUC1 VNTR peptide. We have
cultured the AR20.5 hybridoma in a bioreactor producing large quantities of IgG. Using papain digestion
and cation exchange chromatography we have purified Fab fragments from AR20.5 IgG, for a combination
of binding and structural studies. We have grown crystals of AR20.5 Fab in complex with VNTR peptide
72
POSTER ABSTRACTS
that diffract to 2.2 Å. The Fab structures reveal the nature of the interaction between AR20.5 and cancer
associated MUC1 furthering our understanding of how VNTR peptide glycosylation may influence
binding. Ultimately by understanding epitope recognition and the effects of antigen glycosylation we
seek to improve antibody affinity and specificity for development of the next generation of therapeutics
antibodies. Our results have the potential to have direct translational benefits for the improvement of
treatment outcomes in pancreatic cancer.
POST 10-54
Neutralization of Listeria Monocytogenes by Single Domain Antibodies
2
1
1
2
Ian Huh , Robert Gene , Jyothi Kumaran , Cory Brooks
1
Human Health Therapeutics, Biologics, National Research Council of Canada, Ottawa, Ontario,
Canada, 2California State University, Fresno, Fresno, California, US
Listeriosis is a serious food-borne disease caused by the Gram-positive bacteria Listeria monocytogenes.
The bacteria are of particular concern for pregnant women, as even in asymptomatic cases the bacteria
can cross the placental barrier to cause abortion. Although the bacteria can be treated with antibiotics,
inclusion of a prophylactic that can prevent Listeria colonization as a component of pre-natal care would
greatly reduce the risk of Listeriosis and abortion of the fetus during pregnancy. Bacterial invasion of
epithelial cells is mediated by coupled-bindings of the virulence factors Internalin A (InlA) and Internalin B
(InlB) to receptors on the target cell surface. InlA binds the E-Cadherin while InlB binds the hepatocyte
growth factor receptor (c-Met) to trigger endocytosis of Listeria. Therefore, inhibition of internalin
interactions with their cognate receptors may inhibit Listeria from invading a cell, opening a new
therapeutic avenue. Single domain antibodies (sdAb) are the smallest known antibody fragments that
retain binding function. They are derived from the unique heavy chain antibodies found in camels, alpacas
and llamas. Due to the convex architecture of sdAb binding sites, they can access epitopes unavailable to
conventional antibodies, such as enzyme active sites and protein-protein interaction sites. Using
gentamicin protection assays and flow cytometry we have demonstrated that InlB specific sdAb are
capable of inhibiting Listeria invasion of HeLa cells in vitro. Furthermore, we have discerned the molecular
mechanism behind the capability of the sdAb to inhibit Listeria colonization. We have obtained a high
resolution X-ray crystal structure of InlB in complex with an sdAb. The structure revealed that the sdAb
binds in a negatively charged cavity on the surface of InlB. Comparison of our structure with a structure of
InlB in complex with c-Met revealed that the sdAb directly competes for the same binding site as
the Listeria cell surface receptor, leading to the invasion inhibition. Our results demonstrate the potential
of sdAb as a new class of therapeutics to protect women from Listeria during pregnancy.
POST 10-55
Understanding Regulation of P-Rex1, an Enhancer of Metastatic Potential
1, 2, 3
1, 2, 3
1, 2, 3
Jennifer Cash
, Ellen Davis
, John J. Tesmer
1
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, US, 2Department of Pharmacology,
University of Michigan, Ann Arbor, Michigan, US, 3Department of Biological Chemistry, University of
Michigan, Ann Arbor, Michigan, US
Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guaninenucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer
metastasis. P-Rex1 is synergistically recruited to the cell membrane and activated by PIP3 and
heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell
surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P-Rex1
has thus become an attractive therapeutic target for the suppression of cancer metastasis. However,
development of inhibitors against P-Rex1 is hindered by the fact that its structure and regulatory
73
POSTER ABSTRACTS
mechanisms are poorly understood. My long-term goal is to define the molecular basis for regulation of
P-Rex1 by PIP3 and Gβγ, principally using the technique of X-ray crystallography. The resulting structures
are expected to define the surfaces and residues of P-Rex1 that are important for its interaction with its
regulators, information that will facilitate the rational design of therapeutics that could be used to treat PRex1-associated cancer. Towards this goal, we have determined crystal structures of the Dbl
homology/pleckstrin homology (PH) catalytic core of P-Rex1 in complex with its substrate small GTPases
Cdc42 and Rac1. In addition, we have also determined high-resolution structures for the independent PH
domain in complex with IP4, a soluble analogue of PIP3. These atomic structures have likely defined the
key site responsible for regulation of P-Rex1 by PIP3 and reveal several potential protein−protein
interaction motifs that may serve as Gβγ binding sites or structural elements contributing to autoinhibition
by other domains within the enzyme.
POST 10-56
Expression and Purification of Functionally Active Recombinant Plasmepsin 9 from Plasmodium
falciparum
2, 1
Folasade M. Olajuyigbe
1
Department of Biochemistry, Federal University of Technology Akure, Akure, Nigeria, 2Department of
Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, US
Plasmepsins, a group of homologous aspartic proteinases are attractive drug targets against malaria.
Plasmepsin 9 (PM9) expressed in the blood stage of malaria life cycle with unknown function has been
strongly considered as a potential target. However, recombinant expression of active PM9 for biochemical
and structure-activity analysis of the enzyme has been very challenging. Here, the preliminary report on
the expression and purification of active recombinant plasmepsin 9 fromPlasmodium falciparum is
presented. The plasmid of truncated proenzyme (proPfPM9) from Plasmodium falciparum was expressed
in Escherichia coli Rosetta 2(DE3)pLysS competent cells. The protein was purified from inclusion bodies
using combination of cation exchange and gel filtration chromatography. The expression and purification
fractions were subjected to SDS-PAGE. The zymogen was activated to produce mature form of active
recombinant PfPM9. Catalytic activity test of PfPM9 was determined using a chromogenic substrate, LysPro-Ile-Glu-Phe-Nph*Arg-Leu (RS6). Substrate hydrolysis was examined from substrate cleavage scan.
SDS-PAGE confirmed the expression and purification of truncated proenzyme (proPfPM9) with molecular
weight of approximately 54 kD. Kinetic assays showed that the purified PfPM9 was active. The hydrolytic
activity of PfPM9 on RS6 was confirmed by a shift in absorbance peak from 280 nm to 272 nm at the end
of assay reactions. Inhibition studies on the mature PfPM9 showed that the activity of the enzyme was
inhibited by pepstatin A. Protocols for successful expression and purification of active recombinant
plasmepsin 9 in E. coli have been established in this study. These will aid production of sufficient yield of
PfPM9 for detailed kinetic and structural characterization of the enzyme and development of future
inhibitors against malaria.
POST 10-57
Characterizing HER2 Gene Variation at the Protein Level to Address Racial Disparities in Breast
Cancer Mortality
Wei He1, 4, Matthew Saldana2, Tiffany Scharadin3, Steven Hoang-Phou1, Denise Trans3, Dennis Chang2,
Kermit Carraway2, Paul Henderson3, Matthew A. Coleman5, 1
1
Radiation Oncology, UC Davis, Sacramento, California, US, 2Department of Biochemistry and Molecular
Medicine, UC Davis, Sacramento, California, US, 3Hematology Oncology, UC Davis, Sacramento,
California, US,4NSF Center for Biophotonics, Sacramento, California, US, 5Lawrence Livermore National
Laboratory, Livermore, California, US
74
POSTER ABSTRACTS
African-American women have a much higher rate of breast cancer mortality than Caucasian women.
Notably, African-American women are also found to be at greater risk for more aggressive forms of breast
cancer known as triple negative. Recently, a single nucleotide polymorphism (SNP) of HER2, W452C, which
occurs predominantly in African-American women, was reported significantly correlated with breast
cancer. This study reported that tumors from patients harboring W452C do not amplify the erbb2 (HER2)
gene or overexpress the protein, suggesting that this variant may contribute to breast cancer
development through a novel mechanism. To study the structural and functional differences associated
with W452C, first we looked at over-expression of HER2 harboring W452C mutation in cells, which
resulted in increased disulfide dimer formation, likely caused by the extra cysteine in extracellular domain.
Expression of HER2-W452C also increased basal ATK signaling, which could lead to enhanced
tumorigenecity. To address the biochemical role of the W452C SNP in breast cancer we applied a cell-free
in vitro reconstitution system that uses nanolipoprotein particles (NLPs) to solubilize and support
functional membrane proteins. Cell-free produced wild type HER2 and W452C were tested for tyrosine
phosphorylation as well as specific binding to therapeutic anti-HER2 monoclonal antibodies trastuzumab
and pertuzumab. Our results showed comparable tyrosine phosphorylation levels for both wild type and
W452C HER2, suggesting that the variant itself might not alone be a driver of cancer. We also observed a
decreased binding affinity of trastuzumab for W452C compared to wild type HER2, indicating that W452positive patients might not respond to trastuzumab. On the other hand, W452C had a higher affinity for
pertuzumab. Overall, our studies suggest that the W452C variant may account for a cancer cell phenotype
through a distinct signal pathway. Breast tumors bearing W452C variant may be differentially sensitive to
clinically pertinent therapeutic agents. Further characterization of this variant will be important for the
development of more effective and precise therapeutic intervention treatment regime. This in turn could
lead to better diagnostic decisions to identify the W452C variant and use of targeted measures that better
help address racial disparity problems in breast cancer mortality.
POST 10-58
The Interaction Between the H. Pylori Oncoprotein Caga and Human Pro-Apoptotic ASPP2 is
Distributed Throughout Both Proteins and Involves Intrinsically Disordered Regions
1
2
1
2
1, 3
Tali H. Reingewertz , Anat Iosub-Amir , Daniel A. Bonsor , Assaf Friedler , Eric J. Sundberg
1
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland,
US, 2Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel, 3Microbiology and
Immunology, University of Maryland, School of Medicine, Baltimore, Maryland, US
The leading risk factor for gastric cancer in humans is infection by Helicobacter pylori strains that express
and translocate CagA, which interacts with numerous host cell proteins, dysregulating cellular signaling
and causing transformation of gastric epithelial cells. CagA interacts with the tumor suppressor ASPP2, a
pro-apoptotic protein that specifically stimulates p53-mediated apoptosis, reversing its pro-apoptotic
function and promoting p53 degradation. The structure of a complex between the N-terminal domain of
CagA and a 56-residue fragment of ASPP2, of which only 22 residues were resolved, was recently
described. Here, we present structural and biophysical analyses of the interaction between CagA and
ASPP2. We mapped the binding regions between ASPP2 and CagA using peptide arrays, demonstrating
extensive interactions between CagA and numerous peptides distributed throughout the ASPP2 protein
sequence. Using size exclusion chromatography, as well as circular dichroism and nuclear magnetic
resonance spectroscopy, we found that the ASPP2 region spanning residues 331-692 is intrinsically
disordered in its unbound state. A fragment of ASPP2 within this region, spanning residues 448-692, and
the previously characterized intrinsically disordered proline-rich domain of ASPP2 spanning residues 693918 interact with CagA, which we quantitatively characterized by surface plasmon resonance analysis. We
also observed a weak interaction between CagA and the extreme C-terminal Ank-SH3 domain of ASPP2
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POSTER ABSTRACTS
(residues 893-1128). Additionally, we found that the presence of the C-terminal region of CagA was
dispensable for the interaction with the ASPP2 region spanning residues 331-692, but contributed to the
interaction with the ASPP2 region spanning residues 692-1128. These data implicate numerous previously
uncharacterized regions distributed throughout the protein sequences of both CagA and ASPP2 as
determinants of this protein-protein interaction, providing novel targets for disruption of an important
complex for H. pylori-mediated gastric cancer.
POST 10-59
Is There a Common Structural Basis for Amyloidosis Toxicity? A New Receptor-Mediated
Mechanism of Pancreatic Islet Amyloidosis-Induced Beta-Cell Toxicity in Type 2 Diabetes
Andisheh Abedini1, Annette Plesner3, Ping Cao2, Jinghua Zhang1, Fanling Meng2, Chris T. Middleton4,
Ling-Hsien Tu2, Hui Wang2, Fei Song1, Rosa Rosario1, Martin T. Zanni4, Bruce Verchere3, Daniel P.
Raleigh2, Ann Marie Schmidt1
1
School of Medicine, New York University Medical Center, New York, New York, US, 2Department of
Chemistry, Stony Brook University, Stony Brook, New York, US, 3Child & Family Research Institute and
Department of Surgery and Pathology and Laboratory Medicine, University of British Columbia,
Vancouver, British Columbia, Canada, 4Department of Chemistry, University of Wisconsin-Madison,
Wisconsin, US
Amyloid formation by the hormone islet amyloid polypeptide (IAPP or amylin), causes pancreatic beta-cell
death, consequences of which include diabetes and islet transplant failure. The nature of the toxic species
produced during IAPP amyloid formation and the cellular mechanisms by which it elicits cell death are
unknown. We define the toxic entity produced during IAPP amyloid formation: transient, soluble, loosely
packed, pre-fibrillar, oligomeric lag phase intermediates with modest beta-sheet structure, which appear
to be on-pathway for amyloid formation. The properties of the toxic entities are considerably different
from those proposed for other amyloidogenic proteins, calling into question the hypothesis that toxic
species from different proteins share common features. The toxic intermediates are ligands of the
receptor for advanced glycation end products (RAGE). In a murine model of islet amyloidosis, pancreatic
RAGE protein expression is upregulated and co-localizes with islet amyloid deposits, along with the
apoptosis marker, cleaved caspase 3. Dominant negative inhibition of cellular RAGE via soluble RAGE,
genetic deletion of RAGE or addition of a RAGE-blocking antibody prevents IAPP-induced beta-cell
toxicity. These findings establish that IAPP induced pancreatic beta-cell death is mediated via a novel
receptor-mediated mechanism, and have implications for the treatment of diabetes and for the broader
field of amyloidosis diseases.
POST 10-60
SUMO4 C438T Polymorphism is Associated With Papulopustular Skin Lesion in Korean Patients
with Behçet's Disease
Hyun-Sook Kim
Soonchunhyang University Seoul Hospital, Seoul, Korea
Objects: Small ubiquitin-like modifier 4 (SUMO4) is involved in a range of autoimmune diseases and is
known to down-regulate the transcription activity of nuclear factor kappa B (NF-kB). Our objective was to
investigate the association of a certain polymorphism (C438T) of the SUMO4 gene with Behçet’s disease
(BD) in terms of its incidence and clinical features in Korean patients. Methods: We consecutively enrolled
83 patients with BD and 120 healthy controls. Genomic DNA was extracted from whole blood samples. We
identified a single nucleotide change (C438T) in the SUMO4 gene using an amplification refractory
mutation system (ARMS) technique. To validate the ARMS technique, we compared its results to the
results of direct sequencing in 20 subjects. HLA-B51 status was determined by polymerase chain reaction
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POSTER ABSTRACTS
sequence-specific primers. Results: The presence of papulopustular lesions (p = 0.006) and vascular
involvement (p = 0.045) was significantly different between C438T genotypes in HLA-B51-positive patients
with BD. There were no differences in alleleic or genotypic frequencies of the SUMO4 C438T
polymorphism between patients with BD and controls (p = 0.567 and p = 0.818, respectively). The
difference in papulopustular skin lesions between CC and CT+TT genotypes in HLA-B51-positive patients
with BD was also statistically significant (p = 0.002, OR = 23.40, 95% CI: 2.33 – 235.54). Conclusion: The
C438T polymorphism in the SUMO4 gene is associated with significantly increased risk of papulopustular
skin lesions in HLA-B51-positive patients.
POST 10-61
Calcium-Dependent Proteases and Their Proteinaceous Inhibitor in the Brain Regions Affected by
Neurodegeneration in Rats
Liudmila A. Lysenko1, Nadezda P. Kantserova1, Nikolay L. Rendakov1, Khristina N. Prokopenko2, Nina N.
Nemova1
1
Lab of Environmental Biochemistry, Institute of Biology, Karelian Research Centre of RAS, Petrozavodsk,
Russian Federation, 2Dept of Organic and Biological Chemistry and Molecular Biology, Petrozavodsk
State University, Petrozavodsk, Russian Federation
Neurodegeneration both pathology- and age-associated is followed by a disturbance in protein turnover
and imbalance in main proteolytic systems in nervous tissue. In order to determine the contribution of
intracellular proteases in neurodegeneration we study proteasome, lysosomal, and calpain-calpastatine
systems in animal models of Alzheimer disease and at induced excitotoxicity. There were shown (i) the
ineffectiveness of ubiquitin-proteasome system to control a quality of cellular proteins, (ii) the suppression
in autophagic degradation of damaged proteins, organelles or cells, (iii) the activation of proteases
promoted cell death by apoptosis or necrosis (caspases, calpains). Calpains are calcium-activated
proteases tightly regulated by specific proteinaceous inhibitor, calpastatine. Calpain regulation in
damaged brain regions depends on multiple factors such as (i) calcium dysregulation, (ii) selective calpain
and calpastatine mRNA expression, and (iii) spatial distribution of proteases and their inhibitor. First of all
a rise in intracellular Ca2+ both from intracellular stores and passive currents led to increase in proteolytic
and autocatalytic calpain activity. Secondly the mRNA content of main calpain forms – Capn1 and Capn2 –
is higher while calpastatine level is constant in degenerating brain regions. Thirdly calpains and
calpastatine normally colocalized in neurons are found to be spatially separated in damaged nervous cells.
The resulting effect – deregulated calpain activity – is a common characteristic of degeneration-affected
brain regions; calpain hyperactivation is accounted as a key moment, or point-of-no-return, in
neurodegeneration. However having regard to incomplete information on triggering mechanisms of
neuronal loss and new data obtained on different models of neuropathology (amyloid- and glutamateinduced) we can suppose a phenomenon of calcium signaling deficit on the early stage of pathological
process. In the issue of searching of rational therapy approaches enable to delay neurodegeneration we
evaluate some selective calpain regulators as well as exogenous agents with pleiotropic effects targeted
to stimulate endogenous neuroprotective mechanisms. The work was carried out using technical facilities
of IB KarRC RAS Sharing Equipment Centre and financially supported by RFBR grant 12-04-01597 and the
program «Leader Scientific Schools», progect 1410.2014.4.
POST 10-62
Monomeric IKK: Probing Activation and Specificty
William E. Rogers
Chemistry and Biochemistry, San Diego State University, San Diego, California, US
77
POSTER ABSTRACTS
Inhibitor of KappaB Kinase (IKK) is a major regulation point on the NF-κB activation pathway. Recently we
and other published crystal structures in three states of kinase activation; fully active, partially active, and
inactive. These three structures reveal that the dimer unit of IKK gains increasing distance between the
two kinase domains as it becomes active. This movement occurs within in the structural and dimerization
domain(SDD). From this data we engineered a protein construct that stopped at this point. Here we
investigate how this monomeric version of IKK compares to the the native dimeric, and how this can
provide clues to the kinase's activation and specificity.
POST 10-63
An Ultrasensitive Platform for the Detection of Protein Biomarkers in Spiked Human Serum
Stephen Vance, Marinella Sandros
Nanoscience, University of North Carolina at Greensboro, Greensboro, North Carolina, US
Most biomarkers in the early stage of a disease such as cancer, neurological disorders and atherosclerosis
−16
−12
are present at very low concentrations in serum (10 to 10 M ) amongst a diverse mixture of other
biomolecules. In the case of cancer, apparent symptoms occur after it has metastasized through the body
limiting opportunities for different interventions and therapies. Since the chemical variations that occur
during tumor development can take place over several years, biomarkers could be used to detect cancer
early. The availability of an ultrasensitive detection platform that can profile multiple biomarkers
simultaneously is potentially a powerful method for the diagnosis of diseases and monitoring of
subsequent therapeutic treatments. In the present work, we have developed an ultrasensitive Surface
Plasmon Resonance imaging (SPRi)-based nano-aptasensor for the detection of an inflammatory
biomarker at 5 fg/ml (attomolar) in spiked human serum. This ultrasensitive system was engineered
through the unique integration and combination of the SPRi platform with microwave-assisted surface
chemistry, aptamer technology and nanoparticles, to create a clinically relevant biosensor. The preliminary
results are encouraging and show promise in extending the platform to detect an array of biomarkers in
complex biological fluids that are indicative and even predictive, of disease onset and disease
progression.
POST 10-64
Regulation of Calpain Activity in Fish Brain by Weak Low-Frequency Magnetic Fields
Nadezda P. Kantserova1, Liudmila A. Lysenko1, Natalia V. Ushakova2, Vyacheslav V. Krylov2, Nina N.
Nemova1, 1Institute of Biology, Karelian Research Centre of RAS, Petrozavodsk, Russian Federation, 2I.D.
Papanin Institute for Biology of Inland Waters of RAS, Borok, Yaroslavl' region, Russian Federation
Calpains are calcium-regulated proteases found in the cytosol of all eukaryotic cells; they are involved in
various cellular functions including survival and death. It is known that fish calpain system is similar to that
of mammals and includes two ubiquitous forms (μ- and m-calpains) and their proteinaceous inhibitor,
calpastatin. Dysregulation of the calpain system was found to mediate a variety of pathological conditions
including muscular dystrophy, cancer, inflammation, and neurodegenerative diseases. Nevertheless the
number of therapeutic approaches to regulate calpain activity in vivo through calpain inhibition is limited
due to the low selectivity of synthetic calpain inhibitors and the unsatisfactory pharmacological properties
of developed inhibitors. After a series of experimental works by Liboff and Lednev it was found out that
the immediate target for weak low-frequency magnetic fields in biological systems could be calcium ions.
Because of absolute calcium dependency of calpain-calpastatine system it becomes a suitable model to
study calcium-related mechanism of magnetic field effects on living organisms. In order to demonstrate
how weak low-frequency magnetic fields affect calpains on fish brain there was a series of in vivo and in
vitro experiments carried out. It has been found that intravital effect of indicated factor led to a significant
decrease in calpain activity in fish brain. It was also shown that purified enzymes have also been
78
POSTER ABSTRACTS
substantially inactivated under the effect of weak low-frequency magnetic fields. The results (i) contribute
in our understanding the phenomena of magnetic field effects on living matter, (ii) add the new
information on calpain proteolytic system biology, and (iii) support fundamental basis for the
development of the alternative (non-invasive and non-pharmaceutical) methods targeting calpain activity.
This work was carried out using IB KarRC RAS Sharing Equipment Centre facilities and financially
supported by RFBR grant No. 12-04-01597 and the program «Leader Scientific Schools» (No.
1410.2014.4).
POST 10-65
Amyloid Beta Peptide Aβ40 and Aβ42 Form Separate Fibrils in Binary Mixtures
Xiaoting Yang1, Risto Cukalevski1, Georg Meisl2, Birgitta Frohm1, Tuomas Knowles2, Sara Linse1
1
Biochemistry and Structural Biology, Lund University, Lund, Sweden, 2Department of Chemistry,
University of Cambridge, Cambridge, United Kingdom
Many studies show that the aggregation of amyloid beta (Aβ) peptides lies behind Alzheimer’s disease.
Several isoforms of Aβ have been identified. Aβ40 and Aβ42 are the main species of Aβ peptide and
confirmed to co-exist in the extracellular amyloid plaques. Amyloid fibril formation is a highly specific
event which is very unlikely to form mixed fibrils. Previous studies have found that the aggregation of
pure Aβ peptides displays one transition. This study indicates aggregation of equimolar mixtures of Aβ42
and Aβ40 has two transitions. Aggregation kinetics is followed by thioflavin T (ThT) assay and circular
dichroism spectroscopy. Mass spectrometry is used to detect the Aβ42/Aβ40 monomer ratio as a function
of aggregation time using isotope labels and trypsin digestion. The Aβ42/Aβ40 monomer ratio decreases
from1 at t0 to nearly 0 at the end of the first transition which indicates the first transition is mainly the
aggregation of Aβ42. Complete consumption of both Aβ42 and Aβ40 at the end of the second transition
implies this transition is mainly the aggregation of Aβ40. This is supported by the short half-time, lower
ThT fluorescence intensity of Aβ42 and longer half-time, higher ThT fluorescence intensity of Aβ40 when
aggregating separately. Fibril morphology and node-to-node distance are studied by cryo-transmission
electron microscopy. Fibrils that formed after the first transition display similar morphology to Aβ42 while
after the second transitions it shows co-existence of Aβ42 and Aβ40 fibrils. By comparing the aggregation
kinetics of pure peptides, Aβ42 is found to accelerate Aβ40 aggregation in a concentration-dependent
way, while Aβ40 has a slight retardation effect on Aβ42. Aggregation of each peptide can be catalyzed by
low concentrations of the fibrils formed from the same Aβ peptides by enhancing secondary nucleation.
However, no catalysis is observed when Aβ42 fibrils are added at low concentration to Aβ40 monomer
or vice versa. Surface catalysis and elongation are thus specific events and microscopic processes of the
aggregation in the mixture are mainly affected during the primary nucleation. Aβ42 and Aβ40 do not form
joint fibrils and the mechanism is related to more favorable interactions in the highly organized in register
packing in pure fibrils that dominate over the unfavorable de-mixing entropy.
POST 10-66
Molecular Engineering of L-Asparaginases as Therapeutic Enzymes for the Treatment of Leukemia
Manfred W. Konrad, Christos S. Karamitros
Enzyme Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany
L-asparaginases (L-ASNases) catalyze the deamidation of the amino acid L-asparagine (L-Asn) to Laspartate (L-Asp) and ammonia. Bacterial L-ASNases are approved therapeutic enzymes for use in the
treatment of various blood cancers to deplete serum L-Asn levels. Their efficiency as protein drugs is
based on the fact that several hematological malignancies depend for growth on the extracellular supply
of the non-essential amino acid L-Asn. To avoid the immune response and other side reactions inherent
to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. One human
79
POSTER ABSTRACTS
isoform, hASNase3, belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily where the protein
is synthesized as a single polypeptide chain that is devoid of catalytic activity. Increased expression of
hASNase3 was observed in several tumors, but its functional relevance is unknown. Autoproteolytic
cleavage of this protein into two tightly associated subunits α and β releases the catalytically critical amino
group of Thr168 at the N-terminus of the β-subunit. Recombinant hASNase3 purifies as the uncleaved,
ASNase-inactive form, and undergoes self-cleavage at a very slow rate. We have found that the free
amino acid glycine very selectively acts to accelerate intramolecular processing of hASNase3 both in
vitro and in human cells. The dependence of hASNase3 activation on glycine may be related to the altered
metabolic profile of cancer cells.
The search for biochemical strategies to increase the serum half-life of the enzyme has prompted
considerable efforts to enhance the stability of L-ASNase, by protecting it through e.g. polyethyleneglycol
modification, and packaging into nanocapsules. Encapsulation enhances stability and potentially prevents
exposure of the enzyme to the immune system. We capitalized on the use of the Layer-by-Layer (LbL)
method of biocompatible microcapsule formation, using calcium carbonate particles as core templates for
protein adsorption, which subsequently were coated with poly-L-arginine and dextran sulfate layers. Our
work suggests that efficient encapsulation in combination with successful enzyme engineering will set the
basis for novel ways to treat blood cancers.
POST 10-67
Tweaking the Spines of Kinase Structures
Lalima G. Ahuja1, Jiancheng Hu3, Alexandr P. Kornev1, 5, Andrey S. Shaw3, 4, Susan S. Taylor1, 5, 2
1
Pharmacology, University of California San Diego, La Jolla, California, US, 2Chemistry and Biochemistry,
University of California San Diego, La Jolla, California, US, 3Pathology and Immunology, Washington
University School of Medicine, St. Louis, Missouri, US, 4Howard Hughes Medical Institute, St. Louis,
Missouri, US, 5Howard Hughes Medical Institute, La Jolla, California, US
The Protein kinase superfamily controls signaling pathways by phosphorylating downstream proteins. The
expertise of these enzymes lies in their ability to work as an ON/OFF molecular switches to regulate signal
transduction depending on the need of the cell. Perturbations in this switch are implicated in various
cancers and diseases. Recently, the ON/OFF states of Kinases have been linked to the
assembly/disassembly of spatially conserved hydrophobic motifs at the kinase core called the ‘Spines’.
These are the Regulatory (R) and the Catalytic (C) spines that run through the kinase domain. In the
present study, using the RAFs as a model system, we show how selectively trapping the assembly of R-or
C-spines allows us to lock a kinase in ON or OFF state. These locked activation states serve as tools to
study aspects of kinase mechanism not elucidated so far. The R-spine Phe mutants are constitutively
active and locked in the ON state. Phe mutations in the R-spine promote dynamic assembly and folding
of an active kinase domain such that a BRAF kinase bypasses all regulatory mechanisms required for
activation. These mutants are hence constitutively active and are immune to activation-loop
phosphorylation and dimerization. These mutations hence explain the unregulated activation of RAF
kinases as seen in various cancers. In contrast to the R-spine mutants the C-spine Phe mutants are locked
in the OFF state. These BRAF mutants are ‘pseudo-kinases’ that are unable to catalyze any
phosphotransfer. However, these catalytically-dead kinases are able to activate their cognate partners by
dimerization; elucidating catalysis-independent functions of canonical kinases. These pseudo-kinases bind
their cognate partners as scaffolds and allow for their activation. The C-spine mutants hence explain the
role of dead kinases in the activation of other kinases as seen in some cancers.
80
POSTER ABSTRACTS
POST 10-68
Correlation Between Aβ (1-40) aggregation in E.coli and in vitro Amyloid Fibril Formation
Kalyani Sanagavarapu, Irem Nasir, Celia Cabaleiro-Lago, Sara Linse
Biochemistry and Structural Biology, Lund University, Lund, Sweden
Amyloid fibrils are formed by ordered aggregation of polypeptides and proteins. Many amyloidogenic
proteins are associated with neurodegenerative diseases including Alzheimer’s disease. In bacteria,
inclusion bodies are often formed from the aggregation of overexpressed recombinant proteins. Inclusion
bodies share many characteristics with amyloids. . In fact, similar driving forces are behind the formation
of amyloids and inclusion proteins from partial unfolded proteins QUESTION ADDRESSED Is it possible to
predict the amyloid fibril formation propensity by analyzing the propensity to form inclusion bodies
during expression? METHODS AND MATERIALS A set of 28 single amino acid mutants of Aβ (1-40) were
screened for a change in aggregation behavior on cell cultural level. SDS-PAGE analysis was used to assay
the expression pattern of soluble and insoluble fractions after cell disruption. In vitro fibrillation studies of
seven mutants using thioflavin-T assay were used to investigate how the fibrillation kinetics correlates
with the inclusion body formation. RESULTS A reproducible level of expression of each mutant is achieved.
Expression pattern is greatly variable over the peptide variants. We explore the correlation of inclusion
body formation level with properties such as the peptide net charge and the overall hydrophobicity of the
peptide. The fibrillation kinetics of mutants correlates well with the inclusion body formation. Analysis of
the expression profile allows prediction of fibril formation propensity for mutants that exhibit strong
changes in solubility compared to Aβ (1-40).
POST 10-69
Structure-based Design of Dual Small-Molecule Inhibitors of Mdm2/Mdmx for Efficiently
Reactivating P53 in Cancer Cells
Zhengding Su1, 2, David Duda2, Lingyun Qin1, Yao Chen1, Huashan Zhang1, Weiping Wang1, Brenda
Schulman2
1
Hubei U of Technology, Wuhan, Hubei, China, 2St. Jude Children's Research Hospital , Memphis,
Tennessee, US
Inactivation of p53 functions is an almost universal feature of human cancer cells. Overexpressed
oncoproteins, Mdm2 and MdmX, ultimately impairs p53 function in nearly half of human tumors. By virtue
of its crucial role in cancer cell development, the aberrant interaction of Mdm2/MdmX-p53 represents an
attractive target for cancer therapy. Recent studies have proven that inhibition of both Mdm2 and MdmX
is more efficient than single target to promote cellular apoptosis in cancers. Here, based on segmental
mutational analysis and a high-resolution crystal structure of the N-terminal domain of Mdm2 in complex
with nutlin-3a, we have rationally designed dual small-molecule antagonists of Mmd2/MdmX, with affinity
binding to Mdm2/MdmX in sub-micromolar concentration. One potent dual inhibitor has been confirmed
to enable activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis and growth
inhibition of human tumors. Our work demonstrates the feasibility of rational design of multiple
functional small molecules targeting aberrant protein-protein interactions for cancer therapy.\
POST 10-70
Subtle Differences Between NOS Active Sites Lends Towards the Development of a Bacterial NOS
Specific Inhibitor
Jeffrey K. Holden1, Soosung Kang3, Richard B. Silverman3, Thomas L. Poulos1, 2
1
Molecular Biology and Biochemistry, University of California-Irvine, Irvine, California,
US, 2Pharmaceutical Sciences, University of California-Irvine, Irvine, California, US, 3Departments of
Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois, US
81
POSTER ABSTRACTS
Nitric oxide synthase (NOS) catalyzes the oxidation of L-Arg into nitric oxide (NO) and is found in both
mammals and select bacteria. Previous work by our group and others has established the bacterial NOS as
an excellent antibacterial target for pathogens Staphylococcus aureus and Bacillus anthracis. Since all
currently known NOS’ share a near identical active site architecture (composed of a heme prosthetic
group, substrate binding site and co-substrate binding site) the design of an isoform specific inhibitor
design is not a trivial task. Recently, we designed a series of inhibitors that take advantage of the bacterial
NOS Ile218 and His128 residues; mammalian equivalents are Val and Ser, respectively. From x-ray crystal
structures we have found the binding mode of these inhibitors to both be constrained sterically and
favored by the noncovalent interactions afforded by the bulkier Ile218 and His128 active site residues.
Moreover, kinetic analyses of these inhibitors reveal them to not only be more potent against the
bacterial form of NOS then the mammalian forms but also to have antibiotic-like properties.
POST 10-71
The Role of Extracellular EMMPRIN and its Glycosylation in Modulating Cancerous Phenotypes
Agnieszka A. Kendrick, Elan Z. Eisenmesser
BMG, Univeristy of Colorado Denver, Aurora, Colorado, US
Extracelluar Matrix Metalloproteinase Inducer (EMMPRIN) is a type I transmembrane glycoprotein
implicated in a variety of conditions including: inflammatory diseases, breast, pancreatic, colorectal and
lung cancers. In pancreatic cancer, antibody therapies are now being developed to specifically target
EMMPRIN and subsequently suppress the detrimental effects of EMMPRIN activity in order to increase the
chances of survival for pancreatic cancer patients. Thus far, such therapies have not been fully effective,
presumably due to the lack of information on the molecular details of EMMPRIN activity and its specific
interactions. EMMPRIN is highly upregulated in several different carcinoma tissues and recent data
designates EMMPRIN to be a marker of pancreatic cancer progression. EMMPRIN’s potent stimulation of
both matrix metalloproteinases (MMPs) and cytokines has also been implicated in the progression of
cancer metastasis, specifically by modulating the tumor environment surrounding cancer cells. Another
factor contributing to EMMPRIN’s function is its high level of glycosylation (10-30 kDa). The described
study here aims to directly characterize glycosylated extracellular EMMPRIN in regard to its stimulatory
activity and target interactions, thereby providing basis for the development of potential EMMPRIN
targeting therapies. We have optimized a mammalian expression systems to purify different glycosylation
forms of extracellular EMMPRIN at milligram quantities. Using biological studies we assessed the
difference between unglycosylated and glycosylated extracellular EMMPRIN activity in cancer cell lines.
Additionally, we performed crosslinking pull-down experiments, followed by mass spectrometry to
identify the specific extracellular EMMPRIN interacting partners and we have begun characterizing
potential interactions. Finally, our studies have determined the functional influence of glycosylation on the
activity of extracellular EMMPRIN and begun to unravel the specifics of extracellular EMMPRIN
interactions with other proteins.
82
POSTER ABSTRACTS
POST 10-72
Structural Insights Into the Role of the Smoothened Cysteine Rich Domain in Hedgehog Signalling
Rajashree Rana1, 2, Candace Carroll3, 1, Ho-Jin Lee1, Ju Bao1, Suresh Marada1, Grace Royappa1, Stacey
Ogden1, 2, Jie Zheng1, 2
1
St. Jude Children's Research Hospital , Memphis, Tennessee, US, 2University of Tennessee Health
Science Center, Memphis, Tennessee, US, 3Hardin Simmons University, Abilene, Texas, US
Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled-receptors (GPCRs), and
functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular
cysteine rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its
essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated.
However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the
Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of
the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the
Smo CRDs from bothDrosophila and human. Our results highlight a novel function of the Smo CRD,
demonstrating its role in binding to small molecule modulators.
POST 10-73
CRAMP 16-33 Inhibits the Assembly and Gtpase Activity of Ftsz and Perturbs the Formation of The
Cytokinetic Z-Ring in Bacteria
Shashikant Ray, Dulal Panda
Departement of Biosciences and Bioengineering, Indian Institute of Technology Bombay, India, Mumbai,
Maharashtra, India
Cathelin related antimicrobial peptide (CRAMP), a cationic peptide containing 37 amino acids, is thought
to regulate innate immunity and to provide host defense mechanism in mammals. CRAMP 16-33
(GEKLKKIGQKIKNFFQKL), a part of the CRAMP peptide, has been reported to display antimicrobial
activity.
In this study, we found that CRAMP 16-33 inhibited the growth of B. subtilis and E.
coli cells. CRAMP 16-33 (10 µM) treatment increased the length of bacterial cells by 4.7 folds. Confocal
microscopic images showed that CRAMP 16-33 treatment perturbed the localization of GFP-FtsZ at the
midcell of bacteria. However, it did not damage the membrane structures of B. subtilis cells. In
vitro, CRAMP 16-33 directly interacted with B. subtilis FtsZ and induced conformational changes in FtsZ.
Light scattering and electron microscopic analysis of the assembly of FtsZ indicated that CRAMP 16-33
inhibited the assembly of purified FtsZ. In vitro, the peptide suppressed the GTPase activity of FtsZ but did
not inhibit the binding of GTP to FtsZ. Though CRAMP 16-33 displays sequence similarity with MciZ, a
negative regulator of FtsZ assembly, it could not inhibit the binding of MciZ to FtsZ. Further, CRAMP 1633 did not appear to inhibit tubulin polymerization. The results together suggested that CRAMP 16-33
inhibits bacterial proliferation by inhibiting FtsZ assembly.
POST 10-74
Dimerized Translationally Controlled Tumor Protein (TCTP) induces Interleukin-8 secretion in
human bronchial epithelial cells via MAPK and NF-κB pathway
Heewon Lee, Haejun Pyun, Jeehye Maeng, Kyunglim Lee†
Graduate School of Pharmaceutical Sciences, Colledge of Pharmacy, Ewha Womans University, Seoul,
Korea, Republic of
83
POSTER ABSTRACTS
Histamine-releasing factor (HRF) is being extensively studied, because of its physiological importance to
airway inflammation and tumorigenesis. HRF amplifies allergic reactions by promoting mediator release
from basophils, eosinophils and epithelial cells during late phase reactions. Previously we demonstrated
that NH2-terminal truncated Del-N11TCTP forms dimers through intermolecular disulfide bonds and is a
potent secretagogue of interleukin-8 (IL-8). In this report, using human epithelial cell line BEAS-2B, we
examined the signal transduction events which mediate IL-8 release in response to Del-N11TCTP. We
found that staurosporine (a protein kinase inhibitor), ET-18-OCH3 (a PI-spcific PLC inhibitor), BAY11-7082
(an NF-κB inhibitor), PD98059 (a MEK1/2 inhibitor), SB203580 (a p38 MAPK inhibitor) and SP600125 (a
JNK inhibitor), all blocked Del-N11TCTP induced IL-8 secretion in a dose-dependent manner. We found
from western blot studies that phosphorylation of MEK1/2, MKK3/6, ERK and JNK, as well as, IκBα
degradation, followed by p65 translocation to the nucleus, occurred during treatment of BEAS-2B cells
with Del-N11TCTP. The translocation of p65 subunit was confirmed using confocal microscopy. AP-1
transactivation increased 1.5-fold when BEAS-2B cells were incubated with Del-N11TCTP for 18 hours; and
the transactivation rate of NF-κB was elevated in a dose- and time-dependent manner. Del-N11TCTPinduced IL-8 secretion was inhibited significantly when the cells were treated with p38 MAPK- or NF-κBinhibitors. Based on these results, we conclude that IL-8 secretion induced by dimerized Del-N11TCTP is
regulated by MAPKs and NF-κB in BEAS-2B cells.
POST 10-75
Lysolipids Modulate Aggregation of the Repeat Domain of a Human Functional Amyloid, Pmel17
Zhiping Jiang, Jennifer C. Lee
Laboratory of Molecular Biophysics, National Heart, Lung and Blood Institute , Bethesda, Maryland, US
Pmel17, an essential protein for pigmentation in human skin and eyes, is trafficked to and proteolytically
processed in the melanosome, where it forms fibrous striations to which melanin is deposited.
Previously, we established that the repeat domain (RPT) derived from Mα, the luminal region of Pmel17,
is sufficient to form fibrils under mildy acidic melanosomal conditions. Here, we report the effect of
lipids on RPT aggregation to explore whether intramelanosomal vesicles and/or melanosomal
membrane can initiate and facilitate fibrillogenesis. Membrane mimics, vesicles and micelles, formed
from phospholipids and lysolipids were employed. Along with circular dichroism spectroscopy, we
exploited single-Trp containing variants to gain site-specific information on protein-lipid interaction. We
find that RPT aggregation is strongly influenced and accelerated by the presence of lysolipids, especially
negatively charged lysolipids such as lysophosphatidylglycerol (LPG) whereas two-chained phospholipids
have minimal effects. While LPG stimulates RPT amyloid formation at submicellar concentrations
(optimal LPG-to-protein ratio ~ 15 – 30), LPG micelles decelerate RPT fibril formation. In contrast to
LPG, zwitterionic lysophosphatidylcholine (LPC) promotes RPT aggregation regardless of its
concentration. Our data suggest that the formation of a helical intermediate in LPG by its amyloidogenic
core residues is the molecular basis for LPG modulated RPT aggregation whereas LPC affects RPT
aggregation by forming lipid-protein mixed micelles. Although our data do not preclude the participation
of other factors such as protein-protein interactions on the membrane, the specific protein-lipid
interactions can regulate amyloid formation in vivo as melanosomes are enriched in lysolipids.
84
POSTER ABSTRACTS
POST 10-76
Extracellular Clusterin Suppresses the Formation of Cytotoxic & α-synuclein Species by Interacting
With Prefibrillar Species and Facilitates Their Lysosomal Degradation: Implications in Parkinson’s
Disease
Abdullah Sultan, Bakthisaran Raman, Ch M. Rao, Ramakrishna Tangirala
CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India, Hyderabad, andhra pradesh,
India
Extracellular deposition of misfolded proteins is involved in diseases such as Parkinson’s, Alzheimer’s,
Creutzfeldt-Jakob and dialysis-related amyloidosis. The deposition of these misfolded proteins is
associated with inflammation and local acidosis. Clusterin is an extracellular protein whose levels increase
several-fold during inflammation. In addition to the extracellular form, clusterin also exists in various
intracellular forms. We have investigated the effect of various forms of clusterin on the amyloid fibril
formation of α-synuclein. At neutral pH, extracellular clusterin prevents the amyloid fibril formation of αsynuclein at sub-stoichiometric concentrations. However, intracellular forms of clusterin lack chaperone
activity against α-synuclein amyloidosis. Under conditions of local acidosis (pH 6.0), which occurs at the
sites of inflammation, clusterin exhibits significantly more exposed hydrophobic surfaces and dissociates
into smaller oligomers that show enhanced chaperone activity against α-synuclein amyloidosis. The
amyloid fibrils of α-synuclein are cytotoxic and are resistant to intracellular degradation. Using confocal
live cell imaging and flow cytometry, we found that the species of α-synuclein formed in the presence of
clusterin are not cytotoxic, localize to lysosomes and readily undergo lysosomal degradation. Analytical
ultracentrifugation and dot blot studies revealed that clusterin interacts with the prefibrillar oligomeric
species formed during the course of α-synuclein amyloid fibril formation. Thus, extracellular clusterin is a
potential extracellular chaperone even under conditions of physiological acidosis relevant during
inflammation, with promising therapeutic implications in amyloid-related diseases such as Parkinson’s.
POST 10-77
The Adhesion Regions Of gingipains From P. gingivalis Are Composed Of At Least Two Distinct
Types Of Domains
Charles A. Collyer1, Jinlong Gao2, Daniele Vicari1, Nan Li3
1
Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia, 2Faculty of Dentistry,
University of Sydney, Sydney, New South Wales, Australia, 3School of Chemistry, University of
Wollongong, Wollongong, New South Wales, Australia
Gingipains are multi-domain peptidases that are critical virulence factors expressed by the keystone
periodontal disease pathogen P. gingivalis. The adhesion regions of these proteases are are principally
comprised of a number of modules that belong to the type 1 (T1) family of gingipain adhesins (also
known as cleaved adhesin domains). We have previously reported the first crystal structures of three
homologous variants of these 19 kDa T1 domains and shown that they recognise a number of host target
proteins. We predicted from sequence analysis and binding data that in addition to the T1 domains the
adhesion regions also contain a number of other unrelated adhesins (referred to here as type 2 or T2
gingpain adhesins) and synergistically contribute to the virulence of P. gingivalis. We have recombinantly
expressed and crystallized the first example of an 17.5 kDA T2 adhesin coded for by a fragment of the
gene for the lysine specific gingipain (kgp). We report here the structure refined at 1.05 angstroms
resolution and thereby confirm the existence of the T2 domain family. This structure represents a new fold
family which is distantly similar in topology to that of the plastocyanin/azurin family of proteins but with
no copper binding sites. Like the T1 adhesins it contains a structural binding site for calcium. We observe
that the recombinant T2 adhesin also binds directly to host target proteins but importantly can also
inhibit bacterial invasion in cell based assays. The role of specific binding activity in the cell invasion
85
POSTER ABSTRACTS
mechanism conferred by T2 adhesins is likely to be a critical component the overall virulence of this
pathogen. References [1] N. Li et al. N. Hunter & C. Collyer, Mol Microbiol., 76(4), 861–873 (2010) [2] N. Li
and C. A. Collyer Eur J Microbiol & Immunol., 1, pp. 41–58 (2011) [3] Li, N. et al., N. Hunter & Collyer
C.A. Mol Microbiol., 81(5) 1358-1373 (2011) [4] P. Yun, N. Li, C.A. Collyer & N. Hunter. Infect & Immun., 80
(10) 3733-3741 (2012) [5] Ganuelas, L.A., Li, N., et al., Collyer, C.A. Eur J Microbiol & Immunol., 3 (3), pp.
152–162 (2013)
POST 10-78
Alzheimer’s Disease and Cerebral Amyloid Angiopathy, Doppelgangers?
Rabia Sarroukh2, Ellen Hubin3, 4, 1, Louise C. Serpell5, Nico A. van Nuland3, 4, Kerensa Broersen1, Vincent
Raussens2
1
Nanobiophysics Group, Universiteit Twente, Enschede, Twente, Netherlands, 2Laboratory of Structure
and Function of Biological Membranes, Universite Libre Bruxelles, Brussels, Brussels,
Belgium, 3Structural Biology Brussels, VUB, Brussels, Brussels, Belgium, 4Department of Structural
Biology, VIB, Brussels, Brussels, Belgium, 5School of Life Sciences, University of Sussex, Brighton, Sussex,
United Kingdom
Amyloid-b (Ab) is the component of neuritic plaques in brains of subjects with Alzheimer’s disease (AD)
and Lewy body dementia. Similar plaques are also related to cerebral amyloid angiopathy (CAA) when
coating cerebral blood vessels as well as stated in muscle disorder inclusion body myositis. Ab Plaques are
marshaled in tangles of repeated filamentous entities: fibrils. Connection of distinct fibrils with different
clinical phenotypes has already been clarified for prion diseases through the existence of distinct strains.
Even if recent findings suggest that Ab plaques spread in a prion-like fashion, it is not clear whether
different Ab fibrils may be related to diverse illnesses. About 80% of AD patients also suffer from CAA but
the link between the two diseases remains elusive. Mutations in the APP gene are one of these
connections. We found experimental conditions to produce two different stable polymorphs with the Ab
Italian mutant and demonstrated that AbE22K can form respectively antiparallel and parallel cross-b
fibrils. Further biophysical characterization demonstrates that those two ‘strains’ differ in the accessibility
of the repeating unit. Consequently, they exhibit different predisposition to bind fluorescent probes
(Thioflavin T and bis-ANS). More interestingly, antiparallel fibrils interconvert into parallel ones under
environmental pressure. We suggest that the ability of Ab to populate stable conformations, propagating
distinct structural features and their interconversion may be closely related to the development of CAA
and/or AD. In therapy conceptualization against Ab related illnesses; tropism is an important
phenomenon to be reckoned with.
POST 10-79
Investigating Of The Immunomodulatory Function Ofplasmodium Falciparumhsp70 Expressed
From Various LPS Minus Bacterial Strains
Ofentse J. Pooe2, Gabriele Köllisch3, Holger Heine4, Addmore Shonhai1, 2
1
Biochemistry, School of Mathematics and Natural Science, University of Venda, Thoyandou, South
Africa, 2Biochemistry and Microbiology, University of Zululand, Kwa-Dlangezwa, KZN, South
Africa, 3Parasitology, Philipps University Marburg, Marburg, Germany, 4Division of Innate Immunity,
Priority Area Asthma and Allergies, Research Center Borstel, Borstel, Germany
Heat shock proteins (Hsps) are conserved molecules that constitute a major part of the cell’s molecular
chaperone system (protein folding machinery). Plasmodium falciparum Hsps play an important
cytoprotective role ensuring that the malaria parasite survives under the harsh conditions that prevail in
the host environment. P. falciparum Hsp70-1 (PfHsp70-1) is a ubiquitous, cytosol-localised Hsp70 that is
essential for parasite survival. Apart from their role as molecular chaperones, it is believed that some Hsps
86
POSTER ABSTRACTS
of parasitic origin are capable of modulating host immunity through signal transduction (chaperokine
role). Most investigations focusing on the chaperokine functions of Hsps use recombinant forms of the
proteins produced in E. coli. The main drawback of this expression system is that the produced
recombinant protein co-purifies with lipopolysaccharides (LPS). Although LPS removal techniques have
been developed, they do not completely remove these contaminants, leading to confounding data as LPS
is a strong immune activator. In the current study, we sought to investigate the immunomodulatory
function of PfHsp70-1. The recombinant form of the protein was produced in three bacterial expression
hosts (E. coli XL1 Blue, E. coli ClearColi BL21 and Brevibacillus choshinensis). The protein was expressed
with an N-terminal polyhistidine tag to facilitate purification by nickel affinity chromatography. PfHsp70-1
produced using the E. coli ClearColi andBrevibacillus expression systems was associated with no
detectable traces of LPS. The protein exhibited no immunomodulatory function when it was exposed to
macrophage cells and cultured in vitro. However, PfHsp70-1 expressed in E. coli XL1 Blue was tainted with
LPS contaminants and this protein preparation exhibited immunomodulatory function suggesting that the
LPS background was responsible for the signal. Our findings suggest that PfHsp70-1 does not possess
immunomdulatory
function.
Furthermore
our
study
confirms
that E.
coli ClearColi
and Brevibacillus expression are reliable expression hosts for the production of recombinant protein for
use in immunomodulatory studies.
POST 10-80
Near Infrared Spectral Monitoring Reveals Water Molecular System Dynamics During The
Amyloidogenic Nucleation
Eri Chatani, Yutaro Tsuchisaka, Yuuki Masuda, Roumiana Tsenkova
Kobe University, Kobe, Japan
Amyloid fibrils are protein supramolecular assemblies, the deposition of which is associated with
numerous serious diseases. The formation of amyloid fibrils generally proceeds through nucleation and
growth phases, and the nucleation is one of the most important phases controlling the overall fibrillation
kinetics and final structure of amyloid fibrils formed. However, little is known about the details of how
proteins and their surrounding water molecules vary.
In this study, we have performed in-situ near infrared (NIR) spectral monitoring of the heat-induced
spontaneous fibrillation reaction of human insulin. When absorption spectrum in a region of the first
overtone of water OH bonds over 1300-1600 nm interval was monitored in real time and multivariate
spectral data analyses was applied for unraveling immense information in the spectra, characteristic
spectral changes related to the fibrillation reaction were detected. By combining additional evaluation of
water absorbance pattern called Aquagram, we have newly proposed unique water molecular system
dynamics, i.e., transient dissociation and subsequent development of hydrogen-bonded water networks
during the nucleation phase of fibrillation. The specific transformations of water spectral pattern
observed by the NIR analysis shed light on the role of water molecules in the formation of amyloid fibrils,
and furthermore, could be used as a new biomarker for early non-invasive diagnosis of amyloid-related
diseases.
POST 10-81
Liver Internalization of Cholesterol Decoded by X-rays
Hay Dvir1, 2
1
Biology, Technion - Israel Institute of Technology, Haifa, Israel, 2Cell Biology, La Jolla Institute for Allergy
& Immunology, La Jolla, California, US
Hypercholesterolemia, high levels of plasma LDL-cholesterol, is a major risk factor for atherosclerosis and
coronary heart disease, a leading cause of death in developed countries. The clearance of LDL from the
87
POSTER ABSTRACTS
circulation occurs via endocytosis with the LDL-receptor (LDLR) mainly by liver cells. This hepatic
recruitment of the LDL-LDLR complex to the clathrin-coated pits is mediated by the endocytic adaptor
protein, autosomal recessive hypercholesterolemia (ARH), which binds the NPxY internalization signal at
the cytoplasmic tail of LDLR. Mutations in either the LDLR tail or in ARH lead to familial
hypercholesterolemia and early onset of atherosclerosis. Since the NPxY sequence motif occurs in many
proteins and there are nearly 60 proteins in human that can bind such signals, the apparent exclusive role
of ARH in cholesterol homeostasis has not been understood. We have deciphered this unique
internalization code by crystallographic analysis of the structure of the LDLR-ARH interface.
POST 10-82
RNA Structural Elements and Protein Interactionsthat Regulate HIV Genome Splicing
Blanton S. Tolbert
Chemistry, Case Western Reserve University, Cleveland, Ohio, US
Alternative splicing is a key event of the HIV replication cycle; however, little is known about the RNA
structures and protein interactions that regulate splice site selection. Acceptor site A7 is one of the bettercharacterized sites, where its activity along with donor site D4 is required to remove the Rev Responsive
Element and produce multiply spliced transcripts encoding tat, rev, and nef. The activity of A7 is
suppressed by an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and
activated by an exonic splicing enhancer (ESE3). The hnRNP A1 protein binds the silencer elements to
effectively block ASF-ESE3 interaction, thereby inhibiting A7 usage. Available splicing models assert
hnRNP A1 disrupts RNA secondary structure through 3’-5’ cooperative assembly; however, footprinting
studies show discrete protection of the RNA structure within the hnRNP A1-ssA7 complex. To gain insight
into the molecular mechanisms that regulate ssA7, we have investigated the structural, biophysical, and
biochemical basis of hnRNP A1 recognition of the ISS and ESS3 elements. Here, we present solution NMR
structures of the isolated ISS stem loop (55-nt) and ESS3 stem loop (27-nt). We’ve also used calorimetric
and NMR titrations to determine binding surfaces. Lastly, we present a structural model of the UP1-ESS3
complex that is derived from NMR-STD, mutagenesis, and crystallographic studies. The structural model
offers insight into how hnRNP A1 binds RNA stem loop structures, which is fundamentally different
models used to interpret it’s alternative splicing functions.
POST 10-83
Molecular Mechanism by Which an Intrinsically Disordered Region in Non-Segmented Negative
Strand RNA Virus Phosphoprotein Acts as a Chaperone Of Unassembled Viral Nucleoprotein
Cédric Leyrat, Filip Yabukarski, Malene R. Jensen, Rob Ruigrok, Martin Blackledge, Marc Jamin
Université Grenoble Alpes, Grenoble, France
The genomic RNA of all non-segmented negative-strand RNA viruses (NNV), including numerous human
pathogens (e.g. rabies virus, measles virus, Nipah virus, Ebola virus, …), is condensed by a homopolymer of
nucleoprotein (N), forming long helical ribonucleoprotein complexes named nucleocapsids (NCs) (Figure
1). These NCs are the biologically active templates used for RNA synthesis by the viral polymerase, and
0
thus the replication of these viruses requires the supply of unassembled N molecules (N ) to encapsidate
the progeny RNA molecules. The phosphoprotein (P) is another essential component of the viral
replication machinery of NNV. It forms homo-oligomers and possesses a modular organization with two
stable, well-structured domains concatenated with two intrinsically disordered regions. P connects the
0
0
viral polymerase to NCs and acts as a chaperone of unassembled N (N ) by forming an N -P complex that
prevents nonspecific encapsidation of cellular RNAs. In paramyxoviruses and rhabdoviruses, the Nterminal intrinsically disordered region of P comprises an α-MoRE (Molecular Recognition Element) that is
0
sufficient to keep unassembled N in a soluble RNA-free form (N ). In our recent works, we used a
88
POSTER ABSTRACTS
combination of NMR spectroscopy and SAXS to describe the isolated α-MoRE (Leyrat et al., (2011a)
Protein Science) and full-length P (Leyrat et al., (2012) J. Mol. Biol.) as ensembles of continuously
exchanging conformers that captures their dynamic character. We showed that the α-MoRE has overall
molecular dimensions and a dynamic behavior characteristic of a disordered protein but transiently
populates conformers containing α-helices (Leyrat et al., (2011a) Protein Science). This region of P
0
0
undergoes a disorder-to-order transition upon binding to N , and the crystal structure of the N -P core
complex showed how the MoRE folds upon binding to N and revealed the molecular mechanisms of its
0
chaperone activities. In N , the MoRE of P occupies the binding site that in NCs is occupied by an
exchanging sub-domain from the neighboring N protomer, thus preventing N polymerization and thereby
RNA binding (Leyrat et al., (2011b) PLOS Ptahogens). We will discuss the implications of the dynamics and
of the large conformational space sampled by P in the assembly of new NCs and its role in encapsidating
newly synthesized RNA and the possibility to target this essential complex for blocking viral replication.
POST 10-84
Substrate and Drug Induced Conformational Heterogeneity in CETP
Revathi Sankar, Sanjib Senapati
Indian Institute of Technology Madras, Chennai, India
Background Low levels of High Density Lipoprotein-Cholesterol (HDL-C) are a leading cause of
cardiovascular disease world-wide. Recent studies have shown that Cholesteryl Ester Transfer Protein
(CETP), whose main function is the bidirectional exchange of cholesteryl ester and triglycerides between
HDL and LDL, is an effective target for dyslipidemia. Many small molecule inhibitors have been developed
for CETP, but the molecular mechanism of CETP inhibition still remains elusive. One of the speculations is
that, the inhibitor bound CETP possesses greater affinity for HDL, thus resulting in the formation of a nonproductive HDL-CETP complex. Here, we attempt to explore the mechanism of CETP inhibition by a
comparative analysis of the 3, 5-bis (tri-fluoromethyl) phenyl derivatives class of CETP inhibitors with the
substrate-bound CETP by Protein-Ligand docking and Molecular Dynamics (MD) simulations. Results The
docking results were consistent with the in-vitro experimental IC-50 values and these molecules inhibit
CETP by physically blocking the hydrophobic tunnel. MD results suggested that the inhibitor binding
modulates the overall dynamics of the protein which was validated by the Dynamic Cross Correlation
Analysis and the Principal Component Analysis. The inhibitor bound complexes show greater extent of
twisting at the distal regions of CETP. This twisting facilitates increased exposure of electropositive sidechains, thus improving the protein's affinity towards the highly electronegative surface of HDL. Conclusion
Our work provides a molecular insight on the mode of action of 3, 5-bis (tri-fluoromethyl) phenyl class of
CETP inhibitors. Besides occluding the channel, the inhibitor binding exposes the electropositive sidechains of the protein residues at the N-terminal distal region, thereby increasing the sensitivity towards
HDL, resulting in the formation of a non-productive HDL-CETP complex. The atomistic details gathered
from the simulations would aid in the rational drug discovery of future CETP inhibitors.
POST 10-85
ASB9 N-terminus Plays an Important Role in Creatine Kinase Regulation
Deepa Balasubramaniam2, Jamie Schiffer2, Jonathan Parnell1, 2, Stephan Mir2, Elizabeth Komives2
1
UCSD, La JOlla, California, US, 2Chemistry and Biochemistry, University of California San Diego, La Jolla,
California, US
Creatine kinases (CKs) regulate ATP levels at sites of fluctuating energy demands. Reduced CK activity has
been implicated in diseases such as heart failure and multiple sclerosis, however little is known about CK
regulation. Ankyrin repeat and SOCS box-containing protein, ASB9, (part of an E3 ubiquitin ligase) has
been shown to bind to CK and promote proteasomal degradation of CK. We hypothesize that ASB9 and
89
POSTER ABSTRACTS
its splice variants control the levels and activity of CK at sites where energy is critically needed. We have
focused on the interaction between CKB and splice variant 2 of ASB9 that contains only the ankyrin repeat
domain (ARD). Our results show that the ASB9-ARD binds CKB with very high affinity (nM) and a
stoichiometry of one ASB9 to one CK dimer. Comparison of binding data from an N-terminal truncation
mutant of ASB9 suggests that residues 1-35 of ASB9 contribute to the binding affinity. In addition, the Nterminus of ASB9 seems to be responsible for inhibiting the enzymatic activity of CK when bound to ASB9.
Hydrogen deuterium exchange mass spectrometry (HDXMS) data shows that only one region in CKB
(residues 182 - 203) which is right in front of the active site, is protected upon binding of ASB9 when
comparing the deuterium incorporation of CKB peptides alone and in complex with ASB9. A model of the
CK-ASB9 interaction generated using computational docking agrees with the binding and HDXMS data.
Analysis of the docked model showed that residue D32 in the ASB9 N-terminus binds to the ATP binding
site in CK and probably plays an important role in the CK-ASB9 interaction. Interestingly, a D32A mutant
of ASB9 binds CK, however has reduced affinity and has no effect on CK activity revealing the importance
of this residue. Overall, the N-terminus of ASB9 contributes to the binding of CK, and plays a role in
directly inhibiting the CK activity by altering regions near the active site of the enzyme. We are further
characterizing the N-terminus of ASB9 to narrow down the residues that directly bind CK and determine
the inhibitory kinetic constants of the interaction. The goal of this project is provide essential information
for the development of inhibitors that would interrupt the CK-ASB9 interaction, resulting in increased
amounts of active CK to treat diseases in which there is a an increased energy demand and decreased CK
function.
POST 10-86
Limited Proteolysis And Dissociation Of Trimeric State Of The BRICHOS Domain Increase Its AntiAmyloid Activity
Henrik Biverstål1, 2, Lisa Dolfe1, Erik Hermansson1, Jenny Presto1, Jan Johansson1, 3, 4
1
Center for Alzheimer Research, Karolinska Institutet, Huddinge, Sweden, 2Department of Physical
Organic Chemistry, Latvian Institute of Organic Synthesis, Riga, Riga, Latvia, 3Department of Anatomy,
Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Uppsala,
Sweden, 4Institute of Mathematics and Natural Sciences, Tallinn University, Tallinn, Tallinn, Estonia
The prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray
crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size
exclusion chromatography and electrospray mass spectrometry. A hypothesis is that the trimeric form of
BRICHOS is inactive, and that the putative substrate-binding site then is buried in the subunit interface.
Limited in vitro trypsination of proSP-C BRICHOS takes place exclusively in a large loop between helix 1
and 2., and cleavage in the same region takes place during proSP-C biosynthesis in the ER or Golgi
apparatus. We show here that limited trypsination of BRICHOS results in increased capacity to delay
Aβ42 fibril formation kinetics as measured by thioflavin T flourescence, and that the ability to bind to the
surface of Ab42 fibrils is unaltered after trypsination. Moreover, treatments that increase the dissociation of
the BRICHOS trimer (addition of the dye bis-ANS, known to bind to the putative substrate-binding site, or
detergents) increase the capacity to delay Aβ42 fibril formation, while cross-linking of the BRICHOS trimer
with glutaraldehyde renders it unable to affect Ab42 fibril formation. These results indicate that
modulation of the accessibility of the putative substrate-binding site is a means to regulate BRICHOS
activity during proSP-C biosynhesis, and that it may be possible to enhance the anti-amyloidogenic
activity of BRICHOS by targeting its oligomeric state.
90
POSTER ABSTRACTS
POST 10-87
Thermodynamic And Structural Characterization Of The Binding Of Zn(II) And Other Molecules To
Human Protein DJ-1
Shinya Tashiro1, Jose Caaveiro2, Chun-Xiang Wu3, Quyen Hoang3, Kouhei Tsumoto1, 2, 4
1
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of
Tokyo, Tokyo, Japan, 2Department of Bioengineering, Graduate School of Engineering, The University of
Tokyo, Tokyo, Japan, 3Department of Biochemistry and Molecular Biology and Stark Neurosciences
Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, US, 4Medical Proteomics
Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Parkinson’s disease (PD) is a progressive and devastating neurological disorder for which there is no cure
or effective treatment. Mutations of DJ-1 cause familial PD, although the role of DJ-1 in familial or
sporadic PD remains unresolved. Very recently, three separate reports have demonstrated the binding of
the metal ions Cu(I) and Cu(II) to human DJ-1. This evidence opens new avenues to understanding the
function of DJ-1 and its role in PD. Because the concentration of transition metals is altered in the serum
of PD patients with respect to basal levels, we sought to examine the interaction of metals belonging to
the Irving–Williams series with DJ-1 using high-resolution techniques. An initial screening using
differential scanning fluorimetry and isothermal titration calorimetry reveals that Zn(II) binds to DJ-1 with
great selectivity among the other metals examined: Mn(II), Fe(III), Co(II), Ni(II), and Cu(II). High-resolution
X-ray crystallography (1.18 Å resolution) shows Zn(II) is coordinated to the protein by the key residues
Cys106 and Glu18. The binding of Zn(II) showed sub-micromolar affinity (KD=0.6±0.1 μM) and it is driven
-1
-1
by favorable enthalpy (ΔH=-4.7±0.1 kcal mol ) and entropy (-TΔS=-3.8 kcal mol ). Site-directed
mutagenesis confirms the importance of the residues ligand Cys106 and Glu18. These results suggest that
DJ-1 may be regulated and/or stabilized by Zn(II).
POST 10-88
PICH And BEND3 Form A Complex: Potential Role In The Processing Of Ultrafine Anaphase DNA
Bridges
Ganesha p. Pitchai1, 2
1
Health and medical sciences, The Novo Nordisk Foundation Center for Protein Research, Copenhagen,
Denmark, 2Department of Cellular and Molecular Medicine, University of Copenhagen, Centre for
Healthy Ageing, Copenhagen, Denmark
The human genome undergoes various threats that can lead to genomic instability, a known driver of
cancer and age-associated diseases. Ultra-fine DNA bridges (UFBs) have been identified as one such
threat for genomic stability. Normal, healthy cells can recognize and resolve these bridges, but how cells
achieve this is not fully understood. UFBs are thin, thread like structures connecting the separating sister
DNAs during anaphase, which can be only visualized by staining for PICH (Plk1-interacting checkpoint
helicase) or BLM (Bloom syndrome protein) using antibodies. PICH was identified as a binding partner of
Plk1, and is characterized by its DNA dependent ATPase and dsDNA translocase activity. Knockdown of
PICH induces chromosome non-disjunction resulting in intertwined regions of stretched DNA. Recently
published biophysical data show that the affinity of the PICH-DNA interaction increases under tension,
which may highlight the importance of PICH in the recognition of stretched DNA in UFBs. We recently
identified the BEN domain-containing protein, BEND3, as a new interaction protein partner of PICH, and
we are currently investigating this interaction using various biophysical methods. Our findings show that
PICH and BEND3 directly interact with each other. We are currently mapping the minimal region of
interaction between these proteins in order to identify key residues involved in the interaction, which may
lead to further understanding of PICH-BEND3 complex formation and their role in UFB processing.
91
POSTER ABSTRACTS
Further, we are analyzing the roles of conserved domains in both BEND3 and PICH for their ability to
promote protein:protein or protein:DNA interactions.
POST 10-89
Computational Docking and Site Directed Mutagenesis to Identify Steroid Binding Sites on
Ionotropic Glutamate Receptors
Emily Bartle, Philip Varnes, Carol Parish, Ellis Bell
University of Richmond, Richmond, Virginia, US
AMPA, NMDA, and kainate receptors belong to the ionotropic glutamate (iGluR) family of receptors
located in the post-synaptic neural membrane. These receptors bind glutamate, a major fast excitatory
neurotransmitter, which activates the receptor channels. Consequently, the receptor’s proper function
plays an important role in synaptic plasticity, memory and learning. Mis-regulation of these receptors has
been linked to many neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Therefore,
our research focuses on understanding the receptors’ activity and regulation to ultimately develop
therapeutic applications. The primary aim of the project is to determine the exact locations for the
binding sites of two endogenous neurosteroids, pregnenalone sulfate (PS) and pregnanalone sulfate
(PREGAS), to the receptors. Using molecular docking studies and other computational methods, several
potentially significant residues have been suggested by our collaborator and subsequently tested via sited
directed mutagenesis. Because the site directed mutagenesis has yet to produce significant changes in
binding affinity, we are attempting to answer the same question through alternative methods, such as
limited trypsin proteolysis and hydrogen/deuterium exchange protection assays. This work was supported
by NSF Grant MCB-104995 to EB
POST 10-90
Effects of Agonist and Regulator Binding on the Structure and Conformational Flexibility of the
Ligand Binding Domains of Ionotropic Glutamate Receptors
Forest Barkdoll-Weil, Carol Guzman, Philip Varnes, Ellis Bell
Ionotropic Glutamate receptors (iGluRs) are tetrameric proteins with 4 domains, an amino terminal
domain, the ligand binding domain, the transmembrane ion channel and a largely disordered intracellular
C terminal region. The ligand binding domain binds glutamate, a major fast excitatory neurotransmitter,
which activates the receptor channels. There are three main families of iGluRs, AMPA, NMDA, and kainite
which among other things differ in their responses to two endogenous neurosteroids, pregnenalone
sulfate (PS) and pregnanalone sulfate (PREGAS). To investigate the role that conformational flexibility of
the ligand binding domain plays in response to either glutamate or the neurosteroids (in the presence or
absence of glutamate) we have used Multiwavelength Collisional Quenching (MWCQ) which reports on
the exposure and charge environment of tyrosines and tryptophans in the protein. While Glutamate has
different effects on flexibility depending upon the family of receptor, the use of differently charged
quenching molecules shows glutamate induced changes in the charge environment that can be correlated
with steroid binding ability.
92
POSTER ABSTRACTS
POST 10-91
A Novel CD4-based Chimeric Antigen Receptor for Functional Cure of HIV
Li Liu1, Bhavik Patel1, Mustafa Ghanem1, Zhilli Zheng2, Virgilio Bundoc1, Richard Morgan2, Steven A.
Rosenberg2, Barna Dey1, Edward A. Berger1
1
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases/National Institutes of
Health, Bethesda, Maryland, US, 2Surgery Branch, National Cancer Institute/ National Institutes of
Health, Bethesda, Maryland, US
Development of an effective vaccine against HIV continues to face challenges thwarted by the surfaceexposed trimeric Envelope glycoprotein (Env). Alternate approaches for prevention, eradication or
“functional cure” of HIV are therefore gaining worldwide attention to control spreading of infection, or
elimination of persisting viral reservoirs, or at least prolonged cessation of the administration of HAART
without viral rebound. In an attempt to develop a potent inhibitor of HIV entry, we had previously
designed a bi-functional soluble protein, sCD4-17b, in which the first two extracellular domains of CD4
(the primary receptor for HIV) were attached to the 17b scFv targeting the highly conserved co-receptor
binding site on HIV gp120 protein. Compared to a 10 aa short linker, attachment of the CD4 and 17b scFv
components via a 35 aa long flexible linker exerted extremely high potency and breadth of HIV1neutralization, due to simultaneous binding of both moieties to the same gp120 subunit of the trimeric
Env and thereby preventing Env-mediated HIV entry. Following the success of adoptive transfer of
Chimeric Antigen Receptor (CAR)-modified T cells as a treatment for hematological cancers in clinical
trials, we have utilized the sCD4-17b moiety as the targeting component of a CAR, with the goal of
durable killing of HIV-infected cells as a potential component of a functional cure. We analyzed three
recombinant CAR variants, containing either CD4 alone or sCD4-17b with two different linker lengths (10
aa or 35 aa) as the extracellular antigen-binding domain, followed by sequences that encode a
transmembrane domain and the intracellular CD28 and CD3ζ signaling domains. Our preclinical results
suggest that the targeting moiety CD4-10-17b is superior to CD4-35-17b, consistent with the importance
of serial triggering for CAR potency. Moreover, both CD4-17b CARS were devoid of the undesired activity
seen with the CD4 CAR of rendering CD8 T cells susceptible to HIV infection.
POST 10-92
Some Like it Hot: Determination of Biomolecular Interactions using MicroScale Thermophoresis
Ana Lazic1, Wyatt Strutz1, Nicole Ford1, Stefan Duhr2
1
NanoTemper Technologies Inc., South San Francisco, California, US, 2NanoTemper Technologies GmbH,
Munich, Germany
The analysis of biomolecular interactions is important in the identification of therapeutic targets and
development of diagnostics, as well as providing insights into cellular processes. MicroScale
Thermophoresis (MST), an immobilization-free technology is used to quantitate biomolecular interactions,
ranging from protein-ion to protein-protein affinities. MST, the directed movement of molecules in
optically generated microscopic temperature gradients, is monitored by fluorescence. This thermophoretic
movement is affected by the entropy of the hydration shell around molecules and is highly sensitive to
binding reactions, which affect the size, charge, conformation, and/or hydration shell. We show how MST
can be used to probe interactions with previously unmeasurable targets in close-to-native conditions—in
vesicles/liposomes, cell lysate, or blood serum. In addition, we demonstrate how interactions with proteins
can be quantified in a label-free manner using intrinsic tryptophan fluorescence.
POST 10-93
93
POSTER ABSTRACTS
Effects of Regulator Binding on the Structure and Conformational Flexibility of the Amino Terminal
Domains of Ionotropic Glutamate Receptors
Carlos Metz, Emily Bartle, Alaina Hyde, Ellis Bell
Ionotropic Glutamate receptors (iGluRs) are tetrameric proteins with 4 domains, an amino terminal
domain, the ligand binding domain, the transmembrane ion channel and a largely disordered intracellular
C terminal region. While the ligand binding domain binds glutamate, a major fast excitatory
neurotransmitter, which activates the receptor channels the amino terminal domains of all three bind
endogenous neurosteroids via an unknown mechanism. There are three main families of iGluRs, AMPA,
NMDA, and kainite which among other things differ in their responses to two endogenous neurosteroids,
pregnenalone sulfate (PS) and pregnanalone sulfate (PREGAS). To investigate the role that conformational
flexibility of the amino terminal domains play in response to the neurosteroids we have used
Multiwavelength Collisional Quenching (MWCQ) which reports on the exposure and charge environment
of tyrosines and tryptophans in the protein and gel filtration to assess the impacts of steroid binding on
quaternary structure. Finally CD and thermal melt studies have been used in investigate the effects of
steroids on overall secondary structure and stability.The results provide insight into potential mechanisms
of steroid regulation of ionotropic glutamate receptors.This work was supported by NSF Grants MCB104995 to EB
POST 10-94
The Variant of CREBBP With HAT Domain Deletion Inhibits Hela Cell Proliferation by Downregulating the Expression of TAF1
Zhang Lu-yu1, Li Xin1, Wang Yun-hong1, Wen Quan1, Zhang Jun2
1
Institution of molecular medicine and oncology, Chongqing, China, 2Department of cell biology and
genetics, Chongqing, Chongqing, China
CREB binding protein (CREBBP) is an intrinsic acetyltransferase that plays an important role in many vital
biological processes. This nuclear protein is ubiquitously expressed and involved in the transcriptional
coactivation of many different transcription factors. In the present article, we found that transfection of
HAT domain (histone acetyltransferase domain) deleted CREBBP variant into the cervical cancer Hela cells
could potently inhibit the cell proliferation. It was uncovered that the acetylation of chromatin histone H3
and H4 would drop to a degree accompanied by the knockdown of EP300 phosphorylation after
transfection of the CREBBP-HAT (-) variant. And the expression of TAF1, the largest subunit of the basal
transcription factor TFIID, was also accordingly down-regulated. Obviously CREBBP-HAT (-) with no
histone acetyltransferase activity could competitively interfere with the functions and interactions of
normal CREBBP with other proteins (such as EP300, etc.), which subsequently resulted in decreased
phosphorylation level of EP300 probably by preventing the CREBBP protein from regulating the kinase
expression in MAPK signaling cascade via acetylation-induced reorganization of chromatin. It has been
believed that MAPK/p38 signaling pathway can activate the acetyltransferase activity of EP300 by
phosphorylation in many chromatin-remodeling processes. In turn, further descent of histone acetylation
catalyzed by EP300 might lower the expression of TAF1 and consequently suppress the transcription
activities associated with proliferation in Hela cells. Because, as known, TAF1 generally participates in
active transcription in tumor tissues, serves as coactivators, and functions in promoter recognition and
transcription initiation. Down-regulation of TAF1 could be responsible for the growth inhibition of Hela
cells. The primary investigation suggests an array of new molecular targets relative to transcription
regulation in cervical cancer therapy. Key words: CREBBP; EP300: TAF1; phosphorylation; chromatinremodeling
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POSTER ABSTRACTS
POST 10-95
Effect of Polyethylene Glycol Conjugation on Conformational and Colloidal Stability of a
Monoclonal Antibody Antigen Binding Fragment (Fab’)
Cristopher Roque, Anthony Sheung, Nausheen Rahman, Salvador F. Ausar
Sanofi Pasteur, Toronto, Ontario, Canada
We have investigated the effects of site specific ‘hinge’ polyethylene glycol conjugation (also referred to
as PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen binding fragment
(Fab’) using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet
(UV) absorbance, and fluorescence spectroscopy suggested that the thermal and pH stability of the
secondary and tertiary structures of the Fab’ do not appear to be significantly affected by PEGylation.
Temperature induced aggregation of the Fab’ was evaluated by optical density at 360 nm (OD 360) and
by static light scattering. The results indicated that PEGylation was able to slightly increase the transition
temperature, as well as prevent the formation of visible and sub-visible aggregates. Mechanical stress
results judged by micro-flow imaging (MFI) and measurement of OD 360 showed that the PEGylated Fab’
had significantly higher resistance to surface-induced aggregation compared to the non-PEGylated Fab’.
Analysis of the interaction parameter, kD, for the two Fab’ versions showed highly positive values for the
PEGylated Fab’, indicative of strong repulsive forces among the molecules. In contrast, negative kD values
were observed for the non-PEGylated Fab’, suggestive of attractive forces among the molecules.
Altogether, the results indicate that the conjugation of a Fab’ with PEG induces minor changes in the
structural stability, while improving colloidal stability and resistance to surface-induced aggregation.
POST 10-96
Biochemical And Structural Characterization Of LiaR From Vancomycin-Resistant E. Faecalis: The
‘Master Regulator’ of the Cell-Envelope Stress Response.
Milya Davlieva3, Yiwen Shi3, Michael Zianni5, Troy Johnson1, Paul Leonard1, John Ladbury1, Cesar A.
Arias2, 4, Yousif Shamoo3
1
Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer
Center, Houston, Texas, US, 2Department of Internal Medicine, 5Division of Infectious Diseas University
of Texas Medical School at Houston, Houston, Texas, US, 3Biochemistry and Cell Biology, Rice University ,
Houston, Texas, US, 4Department of Microbiology and Molecular Genetics, The University of Texas
Health Science Center at Houston, Houston, Texas, US, 5Plant-Microbe Genomics Facility, Ohio State
University, Columbus, Ohio, US
The CDC estimates there are over 66,000 enterococcus infections per year in the US (CDC, 2013) and has
classified vancomycin-resistant enterococci (VRE) as a serious threat. In the absence of a good alternative,
the cyclic lipopeptide daptomycin (DAP) has been used “off-label” for the treatment of multidrug-resistant
enterococci infections and has become a key “front-line” antimicrobial against these organisms. The
emergence of daptomycin resistance during therapy has become increasingly frequent and will increase
as DAP is used more broadly. We have shown that DAP resistance in enterococci is linked to mutations in
genes that alter the cell envelope stress response (liaFSR). Mutations to genes encoding the proteins
comprising the liaFSR two-component signaling pathway occur early in adaptation and are a critical first
step toward DAP resistance. LiaR is a ‘response regulator’ in the cell envelope stress response pathway
that regulates several downstream operons essential to adaptation. Among the most critical downstream
regulatory targets of LiaR is LiaX (formally called Yvlb). In this study, we report the expression, purification,
D191N
and biophysical characterization of LiaR and an adaptive variant LiaR
from E. faecalis that is
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POSTER ABSTRACTS
associated with DAP resistance. Using MST to measure the binding of LiaR variants to a ﬂuorescently
labeled DNA duplex containing the predicted binding site of the liaFSR and liaXYZ operons we
demonstrate that the relative affinity of LiaR for the liaFSR operon appears to be much weaker than
to liaXYZ. A putative phosphomimetic mutant of LiaR showed increased affinity for both DNA targets
suggesting that phosphorylation state is a critical component of LiaR specificity. Moreover, the adaptive
D191N
mutant LiaR
enhanced binding of LiaR to the liaFSR and liaXYZ operons. These data suggest that
D191N
LiaR
mimics some of the properties that would be expected of LiaR in an activated (phosphorylated)
state. In order to elucidate the structural basis for LiaR affinity for specific DNA targets, we have solved the
wt
D191N
structure of the DNA binding domain of LiaR and LiaR
alone as well as DNA binding domain of
D191N
LiaR
complexed to double-stranded DNA by X-ray crystallography. Taken together with our
biochemical studies we have elucidated a mechanistic basis for how mutations in LiaR confer increased
resistance to DAP though alterations in signaling.
POST 10-97
Potent Inhibition Of α-Synuclein Fibrillization And Toxicity Through An Energy-Independent
Chaperone-Like Activity
Jan Bieschke1, 2
1
Biomedical Engineering, Washington University in St. Louis, Saint Louis, Missouri, US, 2Max Delbrueck
Center for Molecular Medicine, Berlin, Germany
Alpha-synuclein - a key protein involved in Parkinson's disease (PD) - forms aggregates of amyloid fibrils
that are toxic and cause the loss of dopaminergic neurons. Chaperones, such as heat-shock proteins,
interact with α-synuclein and interfere with aggregate formation, thereby providing a source for potential
neuroprotective intervention. A library of 13,824 proteins was screened in vitro to detect inhibitors of αsynuclein fibrillization in a effort to identify new proteins with anti-amyloid activity, which identified
several potent inhibitors of α-synuclein fibrillization, which were then further characterized. The most
potent modulator, a cytoskeletal linker protein, inhibited α-synuclein fibril formation at sub-stoichiometric
molar ratios of 1:100 and less by an ATP-independent mechanism. Nuclear magnetic resonance (NMR)
spectroscopy and biophysical techniques elucidated that the protein specifically interacts with early
aggregation intermediates, induces the formation of aggregates trapping the N-terminus of α-synuclein
and thus prevents the formation of fibrillization seeds. It significantly reduced α-synuclein-mediated
toxicity in primary neurons, while yeast cells overexpressing mutant α-synuclein confirmed that the
protein rescued α-synuclein-mediated toxicity and aggregation in vivo, suggesting it to be a promising
target for therapeutic intervention.
POST 10-98
Rational Search For A Compound That Selectively Inhibits The Triose Phosphate Isomerase From
Trichomonas Vaginalis
José L. Vique1, Luis G. Brieba2, Rossana Arroyo3, Jaime Ortega3, Arturo Rojo4, Ponciano Garcia4, Claudia
Benítez1
1
Instituto Politecnico Nacional, Mexico, Mexico, 2cinvestav langebio, irapuato, Mexico, 3cinvestav
zacatenco, mexico, Mexico, 4Uam cuajumalpa, Mexico
Trichomonas vaginalis is a protozoan, the causal agent of trichomoniasis, the most common non-viral
sexually transmitted infection (STI) spread worldwide. Trichomoniasis is associated with perinatal
complications and infections in the genitourinary tract in both sexes. For over 40 years, the treatment
against trichomoniasis is the provision of nitroimidazoles, commonly metronidazole and tinidazole.
However, 5 to 20% of the patients show no improvement by this treatment. This highlights the need for
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POSTER ABSTRACTS
new therapeutic regimens against trichomoniasis. Carbohydrates are the main nutrient source
for T. vaginalis. Therefore, the enzymes in the glycolytic pathway on T. vaginalis like triose phosphate
isomerase (TIM) are potential therapeutic targets. We performed molecular interaction simulations
between a set of compounds obtained from libraries and triose phosphate isomerase from T. vaginalis.
Subsequently, the compounds with higher probability of interaction were assayed in their ability to inhibit
or destabilize the mentioned glycolytic enzyme. Some compounds selected by docking strategies were
able to reduce the replication and viability of T. vaginalis cultures. These findings have important
implications in the development of new therapeutic strategies against trichomoniasis.
POST 10-99
The Level Of Dot1L Recruitment Defines The Degree Of MLL-AF9 Hematopoietic Transformation
Aravinda Kuntimaddi, John Bushweller
Molecular Physiology and Biophysics, University of Virginia, Charlottesville, Virginia, US
MLL-AF9 is a chimeric fusion protein arising in mixed lineage leukemia, as a consequence of a
chromosomal rearrangement, and MLL-AF9 leukemias have low cure rates and confer a dismal prognosis.
AF9 interacts with multiple proteins that have transcriptional regulatory roles, and is an intrinsically
disordered protein (IDP), meaning that it is unstructured on its own, but undergoes coupled folding and
binding upon interacting with its partners to form structured complexes. One of the AF9 binding partners
is Dot1L, the only known Histone 3, Lysine 79 (H3K79) methyltransferase. H3K79 di-methylation
(H3K79me2) is widely known to be an epigenetic mark characteristic of transcriptional activation. The
mechanism by which Dot1L is recruited by MLL-AF9 and contributes to dysregulated transcription is
poorly understood. Our goal is to use structure-function studies to fully understand the importance,
mechanism, and the global epigenetic role of the direct recruitment of Dot1L to MLL-AF9. Here we
present the NMR solution structure of the Dot1L-AF9 complex, which forms a mixed alpha-beta structure;
Dot1L forms a beta strand stabilized by three AF9 helices. The interface between the Dot1L and AF9
proteins is largely hydrophobic. Additionally, we show that Dot1L has multiple interacting sites with AF9
that fold similarly upon binding. We generated several structure-guided point mutations on AF9 that
either completely block the Dot1L-AF9 interaction, or have a partial effect by blocking only one Dot1L
binding sites at a time. Introduction of these AF9 mutants into MLL-AF9 hematopoietic progenitor cells
show that they abrogate the in vitro colony forming capacity of MLL-AF9 in a dose-dependent manner,
which is contingent on how many Dot1L binding sites are blocked. To gain an understanding of the global
epigenetic effects of blocking the Dot1L-AF9 interaction, we performed ChIP-Seq analysis of H3K79 diand tri- methylation marks. Our data show that the abrogating the Dot1L interaction with MLL-AF9
mutations leads to large scale losses in H3K79me2 and me3 marks within the HOXA cluster, and that of
early developmental genes, also in a dose-dependent manner. Thus, we present the mechanism of the
Dot1L-AF9 interaction and show that the level of Dot1L recruitment defines the degree of MLL-AF9
hematopoietic transformation.
POST 10-100
Long-range Activity Regulation Mechanisms Within The IL-33/ST2/IL-1RAcp Complex
Kendra Hailey1, Bryan E. Jones2, Patricia A. Jennings1
1
UCSD, La Jolla, California, US, 2Eli Lilly & Company, San Diego, California, US
Interleukin-33 is a dual-function cytokine belonging to the IL-1 family of immune system modulators.
Extracellular IL-33 acts as a modulator of TH2 response by binding the cell surface receptor ST2 and
recruiting a co-receptor, IL-1RAcp. Recent studies identified misregulation of IL-33 can exacerbate breast
cancer growth and anaphylactic shock. Therefore, IL-33 regulation has become an emerging target of
interest for therapeutics. Currently, the only known naturally-occuring inhibitory mechanism is the release
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POSTER ABSTRACTS
of soluble ST2 (ST2L). Therefore, it is critical to have a detailed understanding of how the IL-33/ST2/IL1RAcp ligand-receptor interaction works. Our current approach uses a combination of hydrogendeuterium exchange mass spectrometry (DXMS), receptor-binding assays and in vivo activity assays to
investigate the molecular determinants of novel allosteric regulation within this subgroup of the IL-1
family.
POST 10-101
Modeling and Simulation of Full-length p53 Tetramer Bound to DNA
Ozlem Demir, Pek Ieong
Dpt of Chem & Biochem, University of California-San Diego, La Jolla, California, US
p53 was discovered over 30 years ago, was initially ascribed oncogenic activity, but in 1989 recognized as
what is considered today the most important tumor suppressor protein in humans. A plethora of cellular
and tissue activities are controlled by p53. These processes include induction of cell cycle arrest,
apoptosis, senescence, autophagy, angiogenesis, cell migration, suppression of cancer cell specific
metabolism, and promotion of anti-tumor microenvironments, which collectively are thought to prevent
tumor initiation and maintenance. Understanding the structural details of p53 and its interaction with
DNA is of utmost importance. However, the crystal structure of full-length p53 in its functional tetrameric
form bound to DNA is not available due to its long disordered regions. There are many partial p53
crystal structures available in PDB. Using the recently elucidated structure of DNA-bound tetrameric p53
DNA-binding domains as a template, we have built an all-atom model of DNA-bound full-length p53 in
tetrameric form and explored its dynamics via multiple molecular dynamics(MD) simulations. In addition,
3 different DNA sequences corresponding to p53 target genes that induce different cellular effects are
incorporated into this p53 model and explored by MD simulations. This project will be the analysis of
these MD simulations focusing on the structural and dynamic aspects of p53 and its interaction with
different DNA sequences in atomic detail.
POST 10-102
Putative Programmed Cell Death Pathway Of The Malaria Parasite And The Role Of Cytochrome C
Judith H. Prieto
Chemistry Department, Western Connecticut State University, Danbury, Connecticut, US
Tropical malaria caused by the protozoan Plasmodium falciparum is responsible for more than 200 million
clinical episodes and at least 660,000 human deaths annually. Parasite resistance increases continuously
against the presently available drugs. To further understand the biochemical pathways involved in the
mechanisms of drug resistance, a quantitative proteomic approach has been developed using stable
isotope labeling by amino acids in cell culture (SILAC). In contrast to previous studies with higher drug
concentrations, three different sub-lethal dosage amounts of chloroquine were used to treat parasite
cultures. The protein abundance was quantified and a list of proteins and their related pathways was
collected. Programmed Cell Death (Apoptosis) is one of the control mechanisms used by higher
multicellular animals that are crucial for the continuing development of the surviving cells. Surprisingly the
causative agent of malaria, a protozoan parasite, shows signs of this programmed “suicide”. DNA
fragmentation assays and morphological characterization have been carried out under similar sub-lethal
drug concentrations conditions as the proteome samples pointing to an apoptotic phenotype. The
proteomic data point to some of the players that might be involved. An up-regulation of cytochrome C
suggests it is a protein involved in the apoptosis pathway. We hypothesize that cytochrome C is involved
in a pathway similar to the one found in another unicellular organism, yeast, where after binding a target
protein a proteolytic cascade is turned on. In order to test this working hypothesis an assay was
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POSTER ABSTRACTS
developed to confirm the involvement of cytochrome C in proteolysis of downstream targets using a
fluorogenic substrate.
POST 10-103
Misfolding of the vWF A1 Domain alters the strength of platelet adhesion in Type 2 von Willebrand
Disease
Alexander Tischer, Pranathi Madde, Laurie Moon-Tasson, Matthew Auton
Mayo Clinic, Rochester, Minnesota, US
We have surveyed the effect of a subset of these mutations most frequently identified in vWD patients on
the structure and rheological function of the A1 domain. The mutations in the 2B classification and the
2M classification have a dynamic range of clinical manifestations from a paucity of vWF-platelet
interactions to severe thrombocytopenia. To assess function, we have developed a high-speed video
microscopy analysis of platelet translocation dynamics under shear flow in a parallel plate microfluidic
flow chamber chelated with recombinant A1 domains harboring these mutations. Our analysis of these
dynamics results in statistical distributions of pause (residence) times, proportional to the strength of the
-1
A1-GPIb interaction. At 1500s shear rate, we find a rank order of mean pause times that parallels known
clinical measures of patient vWF activity and platelet counts. Reported patient platelet counts have a
linear correlation to our experimental data indicating that the effect of these mutations on the
conformational properties of the A1 domain accounts for the properties of multimeric vWF. Furthermore,
the severity of thrombocytopenia trends with increasing pause time. To assess structure, we have
developed a number of solution biophysical and thermodynamic metrics that classify these mutational
variants of the A1 domain as Native (with varying thermodynamic stability), Native-Like (having reduced
secondary structure but retaining some thermodynamic stability), and Molten Globule (a complete lack of
tertiary structure with residual secondary structure characterized by the absence of a urea and thermal
unfolding transition). We find that, with the exception of G1324S (Native) and A1437T (Native-Like), all
type 2M mutations induce a molten globule state that abolishes platelet adhesion to the domain. While
many of the type 2B mutations retain native structure, the tendency is to adopt a molten globule structure
that enhances platelet adhesion to the domain. This feature is particularly evident for V1316M and
V1314D that cause severe chronic thrombocytopenia. The results of these studies demonstrate that the
majority of these mutations cause the A1 domain to misfold. Whether the misfolding results in loss or
gain of function depends on the local secondary structure elements involved. These results demonstrate
that subtypes 2M and 2B vWD are categorically protein folding disorders at the A1 domain level.
POST 10-104
Predictive And Experimental Approaches For Characterizing Mutations In Proteins
Maria Teresa Buenavista1, 2, 3, Rohanah Hussain3, Ann-Marie Mallon2, David Nutt4, Liam James McGuffin1
1
School of Biological Sciences, University of Reading, Marlborough, Wiltshire, United
Kingdom, 2BioComputing, Medical Research Council Harwell, Didcot, Oxfordshire, United
Kingdom, 3Beamline B23, Diamond Light Source, Didcot, Oxfordshire, United Kingdom, 4Department of
Chemistry, University of Reading , Reading, United Kingdom
Introduction Our case study is PKD1L1 with a novel point mutation (rks) screened from a mouse model
for ciliopathy. PKD2 and PKD1L1 physically associate in the nodal cilia and this interaction is crucial for
sensing fluid flow that drives subsequent events in the left-right patterning of body organs (sidedness). A
mutation within the second PKD domain of PKD1L1 (PKDd2) is deduced to likely affect its
mechanosensing function and manifest as left-right asymmetry (LRA) disorder. Both predictions and
experiments illuminate on how the differences between the variants may account for reduced mechanical
strength of the PKDd2 domain manifesting as a sidedness disorder. Methods The 3D models of the 83-
99
POSTER ABSTRACTS
residue PKDd2 were generated using the IntFOLD predictive pipeline; they provided the initial structures
for the molecular dynamics simulation simulations. The time-evolved coordinates were studied for their
root-mean-square fluctuations and secondary structures. Steered molecular dynamics (SMD) is currently
being carried out to obtain force and extension measurements. Using synchrotron radiation circular
dichroism, the solution structures of the protein, were compared under three experimental conditions –
normal scan, thermal melt and UV denaturation – for protein secondary structure and protein stability.
The secondary structures were analysed to compare the structural changes between the variants. Results
& Conclusions Under high photon flux UV irradiation, the wild type manifested a slower rate of
denaturation. The thermodynamic and kinetic stability yielded by CD values in the far-UV region correlate
very well with the hypothesis that the mutant has less stable properties. Through MDS, differences in
structural integrity and flexibility of the PKDd2 variants in solution were established. The results showed
that the single amino acid change in the mutant promoted local conformational change evident in the
first 200ns shown as a β-coil transition. The CD spectral signatures confirm the mainly beta composition of
the PKDd2 domain. Likewise, CD results show structural inter-conversions of the domain providing
agreement with models. Computational models and experiments differentiated the structure and
behaviour of the wild type from the mutant – differences which may underpin the impaired
mechanosensing ability of the mutant found in nodal cilia during early development.
POST 10-105
Structural Unity In Diversity In Pilins Of Some Enteric Pathogens
Himadri Biswas, Rajagopal Chattopadhyaya
Biochemistry, Bose Institute, Calcutta, WB, India
The three dimensional structures of four type IVb pilin globular domains in Vibrio cholerae, Salmonella
enteric serovar Typhi, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic Escherichia coli (ETEC)
reported earlier are compared. By superimposing in stereo, it is seen that three of the four pilins possess
five super imposable β-strands, four nearly super imposable α-helices and a disulphide bond, while the
bundle-forming pilin (BfpA) domain of EPEC does not superpose well, exhibiting a different strand
topology, quite different locations for its four helices and several hydrophobic cores instead of a larger
one. As a result, it lacks the extensive side chain-side chain interactions of α1 with almost parallel, three
central β-strands observed in all other type IV pilins. Further, it cannot form the α2-α4 lateral interactions
between consecutive subunits within the left-handed three-start helices forming the fiber. We opine this
structure may not be biologically relevant. Our alternative model for BfpA possesses secondary structures
almost identical to those in the experimental study but is based on the V. cholerae pilin crystal
structure. Structural differences within the type IVb pilins are also discussed.
POST 10-106
Synthesis Of Selectively Functionalized Adiponectin
Andreas Mattern, Annette G. Beck-Sickinger
University of Leipzig, Leipzig, Germany
The adipocyte-derived hormone adiponectin has become a key player for the understanding of
overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. One of its
major functions are the insulin sensitizing effects, which are mediated through the activation of AMPK,
p38-MAPK and PPARα (1). Next to this, it is involved into glucose regulation and fatty acid oxidation.
Recently, three adiponectin receptors AdipoR1, AdipoR2 and T- cadherin have been discovered while an
unknown fourth receptor is hypothesized (2). For only two of them (AdipoR1 and AdipoR2) the signaling
transduction via adiponectin has been confirmed (3). In order to find new binding partners or co-
100
POSTER ABSTRACTS
receptors, we cloned and expressed full length adiponectin as a fusion protein with a C-terminal intein
and a chitin binding domain (CBD) as well as an N-terminal His10-tag. By using the IMPACT-system, the
fusion protein was cleaved to form the corresponding thioester. To separate the starting materials as well
as the cleaved intein chitin binding domain, the purification was performed with chitin beads.
Furthermore, the product was concentrated by Ni-NTA-affinity chromatography. Accordingly, the
obtained adiponectin thioester was reacted with a TAMRA-labeled peptide or a biotin labeled peptide,
respectively, to receive the corresponding ligation product. Finally the functionalized adiponectin was
purified by size exclusion chromatography. Since, the interaction partners for the adiponectin cascade
have not yet been studied in detail, it is important to have a selectively functionalized adiponectin (e.g.
biotinylated or fluorescent labeled). Due to the difficulty of selective protein labeling, this method
provides a good possibility to modify the protein of interest with various functionalities to use it for
further interaction studies. (1) Hui et al. (2012) British Journal of Pharmacology 165, 574–590. (2)
Awazawa et al. (2011) Cell Metabolism 13, 401–412. (3) Heiker et al. (2010) Biol. Chemistry 39, 1005-1018.
POST 10-107
Structural Analysis of the CFA/III Minor Pilin Subunit CofB of Human Enterotoxigenic Escherichia
coli
Hiroya Oki1, Kazuki Kawahara1, Shunsuke Fukakusa1, Takuya Yoshida1, Yuji Kobayashi2, Tooru Taniguchi3,
Takeshi Honda3, Tetsuya Iida3, Shota Nakamura3, Tadayasu Ohkubo1
1
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan, 2Graduate School of
Engineering, Osaka University, Suita, Osaka, Japan, 3Research Institute for Microbial Diseases, Osaka
University, Suita, Osaka, Japan
Enterotoxigenic Escherichia coli (ETEC) is one of the bacterial causes of diarrhea in children and travelers
in developing countries. Adherence of ETEC to host cell surfaces, colonization of the small intestine, and
elaboration of heat-labile and/or heat-stable enterotoxins are essential steps in ETEC pathogenesis. In
human ETEC, the adherence and colonizing abilities depend on the presence of colonization factor
antigens (CFAs), which form pili (or fimbriae) to attach to the target cell surface. In the determined
nucleotide sequence of the whole region for CFA/III formation of human ETEC including a cluster of 14
1
genes, we previously identified the genes encoding major and minor type IVb pilin subunit CofA and
CofB, respectively. To understand the structure and assembly mechanisms of the type IVb pilin subunits,
we recently determined the crystal structure of CofA and discussed the characteristic mechanism of the
2
filament formation of CFA/III pili of human ETEC. However little is known about the functional role of its
minor pilin subunit CofB, which is crucial to understand comprehensively the colonization and adhesive
behavior of human ETEC. Therefore we determined here the crystal structure of CofB at 1.88 Å resolution.
The structure of CofB showed unique three-domain architecture, distinct from that of other type IV pilins.
The central and C-terminal β-sheet-rich domains exhibit no structural homology to any so far
characterized protein fold. The N-terminal domain, on the other hand, forms αβ-fold typical of the type
IVb pilin with remarkable similarity to that of CofA, and consequently fits very well to the previously
proposed filamentous model of CFA/III pili of human ETEC by a simple molecular docking approach. In
this presentation, we will report the details of CofB structure and also discuss the functional mechanisms
of CFA/III pili of human ETEC based on utilizing the available structural information of both its major and
minor pilin subunits. 1. Taniguchi, T., Akeda, Y., Haba, A., Yasuda, Y., Yamamoto, K., Honda, T. &
Tochikubo, K. (2001). Infect Immun 69, 5864-5873. 2. Fukakusa, S., Kawahara, K., Nakamura, S., Iwashita, T.,
Baba, S., Nishimura M., Kobayashi, Y., Honda, T., Iida, T., Taniguchi, T., Ohkubo, T. (2012). Acta Cryst. D68,
1418-1429.
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POST 10-108
Analysis Of The Gene Products Related To Osseointegration In The Early Stage Of Titanium
Implantation
Masataka Horiuchi1, Rumi Horiuchi2, Masanori Ochiai3, Atsuro Yokoyama2
1
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-gun, Hokkaido,
Japan, 2Graduate School of Dental Medicine, Hokkaido University, Sapporo, Hokkaido, Japan, 3Institute
of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido, Japan
Dental implant is one of the effective techniques to recover lost teeth. Although its success rate is more
than 90%, several percent failure is caused by unknown mechanism. In this study, we have set up the
hypothesis that micro-movement caused by the titanium implant induces the expression of genes which
prevent osseointegration. In order to identify the genes expressed by micro-movement, we
comprehensively evaluated by whole genome microarray analysis of the tissues around the titanium
implants in both rat models of acquired and of non-acquired osseointegration. The microarray data
suggested the existence of the group of genes involved in bone formation induced from osseointegration,
while that of the group of genes involved in non-acquired osseointegration. To further investigate the
relationship between those gene products and osseointegration, we are producing Thorombospondin-2
proteins as one of the factors to ensure osseointegration and C1qTNF6 proteins as one of the factors to
prevent osseointegration, by using GRP-tag affinity purification system (Horiuchi et al, Protein Eng Des
Sel., 2012). In future, we will attempt to directly inject these recombinant proteins to the implant region in
the model rats in vivoand validate the effects of those proteins on osseointegration.
POST 10-109
Advanced Molecular Tools for Proteomic Analyses of Microvesicles
Masood Kamali-Moghaddam, Liza Löf, Felipe Oliveir, Lotta Wik, Di Wu, Junhong Yan
Dept. of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
Despite a large number of protein biomarker candidates presented in the literature, only a small group of
proteins have been demonstrated to be clinically useful. Identification of reliable biomarkers in the
biological samples requires access to technologies with sufficient specificity and sensitivity to meet the
complexity of, for instance, blood proteomic. In addition, the focus on proteins as biomarker has now
being expanded to other biomolecules such as high molecular weight microvesicles and exosomes. We
have previously described a sensitive and specific assay (4PLA) for detection of complex target structures
such as exosomes in which the target is first enriched via an immobilized antibody and subsequently
detected by using four other antibodies with attached DNA oligonucleotides. The requirement for
coincident binding by five antibodies to generate an amplifiable reporter DNA molecule results in
increased specificity and sensitivity. Here, we illustrate the application of different formats of proximity
assays for detection and characterization of microvesicles including the surface protein profiling and
analyses of the total protein content using large sets of protein panels
POST 10-110
Bovine Brain Ribonuclease is the Functional Homolog of Human Ribonuclease 1
Chelcie H. Eller2, Jo E. Lomax1, Ronald T. Raines2, 3
1
Cell and Molecular Biology, Univerisity of Wisconsin-Madison, Madison , Wisconsin, US, 2Biochemistry,
University of Wisconsin-Madison, Madison, Wisconsin, US, 3Chemistry, Univerisity of WisconsinMadison, Madison , Wisconsin, US
Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo,
ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells.
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POSTER ABSTRACTS
Understanding these putative roles has relied on continual comparisons of human RNase 1 to bovine
RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different
physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1
and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease,
and find pronounced similarities between that protein and human RNase 1. Specifically, we report that
human RNase 1 and bovine brain ribonuclease have a similar thermostability, activity against single- and
double-stranded RNA substrates, pH–rate profile, affinity for cell-surface glycans, and ability to enter cells.
Our results suggest that brain ribonuclease—not RNase A—is the true bovine homolog of human
RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1
in mammals.
POST 10-111
Defect in the Intramolecular and Intermolecular Cross-linking of Collagen Caused by Hcythiolactone
Marta Rusek
Department of Patophysiology, Medical University of Lublin, Lublin, Poland
Severe hyperhomocysteinemia due to cystathionine-β synthase (CBS) deficiency are associated with
connective tissue (CT) abnormalities in the skeletal system, lung, eye, and vasculature (1), including
atherosclerosis and osteoporosis. Homocysteine (Hcy) is metabolized to Hcy-thiolactone (HTL) in an errorediting pathway catalyzed by methionyl-tRNA synthetase (3) and indirect incorporation into protein by a
post-translational modification. N-Hcy-protein adducts are formed in chemical reaction of HTL with εamino groups of protein lysine residues, called protein N-homocysteinylation (4). It generates modified
proteins and affects in a loss of their enzymatic activities (5). To gain insights into a role of this reaction,
we studied Hcy content of collagen, the main component of connective tissue. Collagen Nhomocysteinylation would block the ε-NH2 groups of lysine residues, which would impairs cross-linking
(6). A reduced number of cross-links was observed in patients with homocystinuria. Because CT are
supramolecular assemblies, even low levels of N-Hcy-lysine in collagen can result in a structural defect.
-/We studied Tg-I278T Cbs mice (tHcy=272±50 μM, N-Hcy-protein=16.6±4.1 μM), and Tg-I278T
+/+
Cbs mice (tHcy=1.9±1.6 μM, N-Hcy-protein=1.6±0.3 μM) (7). We prepared collagen from bones of
these mice using the acetic acid extraction method and analyzed its N-Hcy-collagen content by HPLC
-/method. In a Tg-I278T Cbs mouse model, bone N-Hcy-collagen is significantly elevated compared with
+/+
Tg-I278T Cbs animals (61.0±41.2 vs. 18.3±5.7 pmol/mg bone, P=0.042. Collagen pyridinoline cross-links
-/are significantly decreased in Tg-I278T Cbs mice compared with wild type littermates (20.8±3.5 vs.
15.2±2.7 pmol/mg bone, P=0.022). These findings demonstrate that HTL prevents cross-linking of
collagen, which is a target for N-homocysteinylation in vivo in mice and can account for CT abnormalities
observed in severe hyperhomocysteinemia. References: 1. Mudd et al. Am J Hum Genet. 1985; 37(1):131. 2. Hamelet et al. Exp Mol Pathol. 2007; 83(2):249-253. 3. Jakubowski, J Biol Chem. 1997;272(3):19351941. 4. Jakubowski, FASEB J. 1999;13(15):2277-2283. 5. Jakubowski, Cell. Mol. Life Sci. 2004;61(4):470-487.
6. Liu et al. J Biol Chem. 1997;272(51):32370-323777. 7. Jakubowski et al. FASEB J. 2009;23(6):1721-1727.
POST 10-112
Thermodynamic Analysis of Membrane Interactions with Tau Peptides
Megan Culp, Larry Masterson
Hamline University, Lno Lakes, Minnesota, US
The protein tau is abundant in neurons and can become misfolded, which is a hallmark for Alzheimer’s
disease. Normally tau stabilizes the cytoskeletal structure through a region in tau called the microtubule
binding domain (MBD). The MBD contains four repeat segments referred to as R1, R2, R3 and R4, and
103
POSTER ABSTRACTS
variability in the combination of these segments defines the specific tau isoform. During Alzheimer’s
disease tau is misfolded, leading to the aggregation of these repeat segments within the neuronal cell
body. Because the fold of a protein dictates its function, a protein that is misfolded will result in a lack of
function or a complete loss in its functionality. Previous studies have found that aggregates of tau are
localized to the Golgi membrane, but not at the plasma membrane. Other studies have indicated that the
introduction of membrane mimics can induce tau aggregation. In this work, the membrane affinity for
each repeat segment in tau will be examined by using model membranes of the Golgi and plasma
membranes. The thermodynamics of this binding event will be investigated by isothermal calorimetry in
order to evaluate the thermodynamic contributions upon binding. In addition to the sequence
dependence of the repeat fragments on membrane binding, temperature dependent studies will also be
performed to help examine the role of solvent exposure of polar and nonpolar residues.
POST 10-113
Molecular Basis Of Heme Capture By Isd System Of Staphylococcus Aureus
Jose M. Caaveiro, Nhuan Vu, Koldo Morante, Yoshitaka Moriwaki, Ryota Abe, Kouhei Tsumoto
The University of Tokyo, Minato-ku, Tokyo, Japan
The iron-regulated surface determinant (Isd) is composed by more than ten extracellular and membrane
proteins that adquire heme for the human pathogen Staphylococcus aureus. Isd is an attractive target to
battle S. aureus because its inactivation would interfere with the ability of this pathogen to grow during
bacterial infection. Two key questions relevant to the mechanism of Isd proteins pursued in our laboratory
are (1) how Isd receptors extract the heme from human hemoglobin, and (2) what are the basis of heme
transfer between Isd proteins. We have addressed these questions from structural, biochemical and
biophysical standpoints. With respect to the first question, we report that IsdH-NEAT3 (the first
extracellular heme-receptor of the Isd system) binds particular types of metal-porphyrins displaying
metals in oxidation state (III) but not in oxidation state (II). This observation may explain the susceptibility
of S. aureus, as well as other pathogenic microorganisms, to a novel class of antimicrobial compounds
based on a protoporphyrin IX scaffold. With respect to the second question, we have investigated the
mechanism by which heme is transferred between Isd transporters. So far this characterization has been
frustrated because of the transient nature of the protein-protein interactions involved. A genetically
encoded photoreactive probe was used to survey the regions of IsdC involved in hetero- and selfdimerization. We show that the IsdC transporter not only passes heme to other Isd transporters, as
previously described, but performs self-transfer reactions explaining its central role in Isd system. In
addition, we identified structural elements required for the rapid and specific transfer of heme between
the couple of transporters IsdC and IsdE. We conclude that the ultra-weak interactions between Isd
transporters are governed by bona fide protein structural motifs.
POST 10-114
Biologically Responsive Recombinant Protein Anchors For Macromolecular Drug Delivery
Jason S. Buhrman1, 2, Jamie E. Rayahin1, Yu Zhang1, Mary Tang1, Richard A. Gemeinhart1, 3, 4
1
Biopharmaceutical Sciences, University of Illinois, Chicago, Illinois, US, 2Medical Scientist Training
Program, University of Illinois, Chicago, Illinois, US, 3Bioengineering, University of Illinois, Chicago,
Illinois, US,4Opthalmology and Visual Sciences, University of Illinois, Chicago, Illinois, US
Therapeutic macromolecules present unique delivery challenges that have historically limited their clinical
utility. Exogenous macromolecules tend to be unstable in the body and therapeutic doses commonly do
not reach disease targets. Polymeric delivery systems have shown great success in protecting
macromolecular cargo thereby increasing their duration in the body. Release mechanisms that dissociate
macromolecular cargo from a delivery vehicle have largely been based on degradation of the vehicle
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POSTER ABSTRACTS
and/or simple diffusion of the cargo from the polymer system. Ideally, cargo would only be released in
the site of disease that would require lower doses of the macromolecule and would minimize off target
effects. We sought to create an anchor system that would immobilize macromolecular therapeutics to a
delivery vehicle until they reached a disease site. We used the natural affinity between glutathione Stransferase (GST) and glutathione (GSH) to immobilize recombinant protein and DNA via DNA-binding
protein to the surface of poly (ethylene-glycol) diacrylate (PEGDA) microspheres. The anchor was stable
for six days in simulated physiologic fluid matching extracellular conditions and for 48 hours after intracerebral implant. Functional macromolecular cargo could be released from the surface of the PEGDA
microspheres by enzyme catalyzed proteolysis, and by increased glutathione levels. Environments with
activated proteolytic enzymes or high GSH concentrations are common in several disease states including
bacterema, cancer, wound healing, and venous thrombosis. Due to the ease of creation, and the already
high numbers of therapeutic proteins being purified with GST fusion anchors, we believe the GST/GSH
anchor to be a promising component of future protein delivery systems.
POST 10-115
X-ray Crystallography & Small Angle X-ray Scattering Studies of Interferon Regulatory Factor 4
Soumya Govinda Remesh, Carlos R. Escalante
Virginia Commonwealth University, Richmond, Virginia, US
Transcriptional regulation involves the orchestrated binding of transcription factors to regulatory DNA
binding sites known as enhancers. Interferon (IFN) regulatory factor family member (IRF4) is a
transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes, is
limited to the immune system and is critical in B- & T-cell differentiation. Generally, members of IRF
family, like IRF3 and IRF5, have carboxy terminal auto-inhibitory regions that are phosphorylated to
generate a transcriptionally active dimer. IRF4 also has a carboxy terminal auto-inhibitory region and it
gets activated by binding to multiple different partners presumably through a general mechanism of
activation with slight variations with each binding partner. For my graduate work, I am investigating the
general mechanism of activation of the auto-inhibited IRF4 to compare it with other members of the IRF
family. I have determined the crystal structure of the C-terminal activation domain of IRF4 and carried out
small angle X-ray scattering (SAXS) studies to generate ab initio models for the full-length protein and
obtain insights into the autoinhibitory mechanism. The data suggests that the putative linker of IRF4
connecting the N- and C-terminus is most likely a folded well-structured domain that interacts with the
auto-inhibitory carboxy tail. I am currently working towards structurally characterizing the putative linker
region as well using X-ray crystallography, circular dichroism and analytical ultracentrifugation.
Information about the mechanism of activation of the auto-inhibited IRF4 highlighting its uniqueness
within the IRF family will help in development of novel therapeutics in disease states that are mediated by
IRF4 like multiple myeloma, cardiac hypertrophy and certain autoimmune diseases.
POST 10-116
Structural Characterization of Protein Aggregates of Wild-Type and Disease Associated Variants of
Human γS -crystallin
David M. Montelongo, Chelsea Anorma, Diana Bandak, Rachel W. Martin
Department of Chemistry, University of California, Irvine, Irvine, California, US
Protein aggregation has been implicated in numerous diseases as well as in vital cellular processes in
organisms ranging from bacteria to humans. Proteins can form aggregates via differing mechanisms
according to the folding state of the monomeric building block of the aggregate, which can include
native-like structures or alternate stable states. γ-crystallins are a family of structural proteins that
maintain the refractive index gradient of the eye lens, and aggregation of these proteins results in cataract
105
POSTER ABSTRACTS
formation. Any of several known point mutations in the primary amino acid sequence of the structural
crystallins can result in hereditary early-onset cataract formation, with many examples known for γDcrystallin and the the G18V variant of γS-crystallin. In this study, concentrated samples of wild-type γScrystallin as well as G18V and G106V variants were subjected to a range of solution conditions, which
included varying pH (pH 2, 4, 7, 9) and temperature (22°C or 37°C). Each variant produced aggregates
under at least one of the conditions tested, with the G18V variant showing increased aggregation
propensity compared to the wild-type and G106V variants. Each sample was analyzed using a Thioflavin T
fluorescence assay to detect the presence of amyloid-like aggregates against a lysozyme fibril positive
control. Thioflavin T fluorescence was detected in wild-type and G106V samples only at elevated
temperatures and acidic conditions. Fluorescence was detected in the G18V variant at both temperatures
tested and under both acidic and basic conditions, suggesting an increased propensity for this variant to
form amyloid fibril structures. Fluorescence was not present in every aggregate sample, suggesting that
γS-crystallin may have more than one stable state that results in aggregate formation, and that the
protein can form both amyloid and non-amyloid aggregates. The presence or absence of amyloid
formation in each sample was verified by imaging techniques, congo red staining, and x-ray powder
diffraction. Efforts to understand the structures of these aggregates on a molecular level are also being
undertaken with the use of solid-state NMR techniques on isotopically labeled protein aggregate samples.
POST 10-117
Insight Into the Catalytic Mechanism of GABA-producing Enzyme: Glutamate Decarboxylase from
Sphaerobacter thermophilus
Ruiying Wu1, Shonda Clancy1, Andrzej Joachimiak1, 2
1
Midwest Center for Structural Genomics, Biosciences, Argonne National Laboratory, Argonne, Illinois,
US, 2Structural Biology Center, Biosciences , Argonne National Laboratory, Argonne, Illinois, US
Glutamate decarboxylase (GAD; EC 4.1.1.15) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme, which
catalyzes the irreversible a-decarboxylation of L-glutamate to g-aminobutyrate (GABA). This enzyme is
widely distributed amongst eukaryotes and prokaryotes. The GAD plays a crucial role in the vertebrate
central nervous system where it is responsible for the synthesis of GABA, a major inhibitory
neurotransmitter. In the current study, we have determined at atomic resolution several crystal structures
of GAD from Sphaerobacter thermophilus (StGAD), the apo-form, several mutants and in complex with its
cofactor PLP, substrate and product. StGAD has shown high structure similarity with two isoforms of
human GAD67 and GAD65. The enzyme contains a flexible loop near the active site. The location of the
residues 330-340 in the loop has shown significant difference as observed in wild type and that in three
mutant Y333A, Y333Q and Y333N, especially the residues of Tyr333, Met334 and Arg335. Both Lglutamate (substrate) and GABA (product) are found near the PLP-binding domain and the mobile loop,
respectively, in the Y333A mutant StGAD structure. The findings reported herein provide evidence for
Y333 as the critical residue responsible for the inherent mobility of the catalytic loop and suggest a key
role in the catalytic reaction. The accurate molecular detail of StGAD interacting with its substrate,
product as well as its PLP cofactor in the atomic structure provides insight into the one-step GABA
biosynthesis catalyzed by the glutamate decarboxylase. Detailed information will be presented.
This
work was supported by National Institutes of Health Grant GM094585 and by the U.S. Department of
Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.
POST 10-118
Interactions of E. coli Immunoglobulin Binding Protein D and the Fc Part of IgG
Kornelia M. Mikula1, 2, Robert Kolodziejczyk3, Adrian Goldman1, 3
106
POSTER ABSTRACTS
1
Department of Biochemistry, University of Helsinki, Helsinki, Finland, 2The National Doctoral Program in
Informational and Structural Biology, Åbo Academy, Turku, Finland, 3Astbury Centre for Structural
Molecular Biology, University of Leeds, Leeds, United Kingdom
The discovery of penicillin started antibiotic era, the turning point in the history of medicine. Since then,
finding new and more effective drugs has been at the forefront of biomedical research to combat
infectious diseases. Bacteria, however, simultaneously have mutated and gained resistance to them. Even
though scientists have eliminated some deathly diseases, now bacteria possessing multidrug resistance
are again threating humanity. New ways of combating bacteria need to be invented, meaning that our
understanding of pathogen-host interactions has to be improved. During infection, gram-negative
bacteria express variety of virulence factors that interact with the host organism enabling their survival
and progression of host invasion. Among the first and highly expressed are trimeric autotransporter
adhesins (TAAs). TAAs comprise large protein family that includes Escherichia coli immunoglobulinbinding proteins (Eibs), such as EibA, C, D, E, F, and G (1, 2). They bind human immunoglobulins and
increase serum resistance (3). Additionally, EibD is an autoagglutinin and promotes biofilm formation (1).
It is believed that binding of immunoglobulins by bacterial surface proteins allows E. coli to escape host
immune surveillance and promote infection (4). Here, we present studies of interactions between EibD and
the Fc part of immunoglobulin G. Based on double mutant binding studies, we have identified key amino
acid residues involved in EibD - Fc interactions and modeled structure of the EibD - Fc complex. (1) C.H.
Sandt, C. W. Hill, Infect Immun 68, 2205-14 (2000). (2) Y. Lu, S. Iyoda, H. Satou, K. Itoh, T. Saitoh, H.
Watanabe, Infect Immun 74, 5747-55 (2006). (3) J. C. Leo, A. Goldman, Mol Immunol 46, 1860-66 (2009).
(4) J. D. Lambris, D. Ricklin, B. V. Geisbrecht, Nat Rev Microbiol 6(2), 132-142 (2006).
POST 10-119
The Dynamic Functional Consequences of the Thrombin-Thrombomodulin Interaction
Lindsey D. Handley, Elizabeth A. Komives
UC San Diego, San Diego, California, US
The coagulation cascade is critical to wound healing, but must be well regulated to prevent pathological
clot formation. We previously investigated the backbone dynamics of a key component in this cascade,
thrombin, and found that the loops surrounding the thrombin active site show dynamics on the ps-ns
timescale when the active site is inhibitor-bound, but have dynamics on the μs-ms timescale when the
active site is unoccupied. Thrombomodulin (TM) plays a key role in the regulation of the coagulation
cascade by switching the function of thrombin from procoagulant to anticoagulant. A recent molecular
dynamics study suggests TM does this by influencing the slow timescale dynamics of thrombin, but the
actual timescale of these motions is currently unknown. NMR measurements of chemical shift differences
(CSDs) can be used to probe changes in the conformational ensemble of thrombin upon TM binding.
Other NMR experiments probe the timescales of motion, including R1 and R2 relaxation experiments,
which probe the ps-ns time regime, and TROSY Hahn Echo and CPMG experiments, which probe slow (μsms) timescale motions. To perform these NMR studies, our lab has developed a truncation of human TM
15
(TM456m) with complete thrombin-altering activity. We have produced N-labeled human thrombin
15
15
using an E. coli expression system and have collected HSQCs of unbound N-thrombin and N-thrombin
in 1:1 complex with unlabeled TM456m. Binding of TM456m induces significant CSDs and differences in
peak intensities throughout thrombin. Several residues in the TM binding site, including S36A, L65, I82,
M84, and K110, become significantly perturbed upon binding. TM binding also causes significant CSDs in
several residues near the active site, including H57 and D102 of the catalytic triad. We hope that a
complete understanding of how TM alters the dynamics and function of thrombin will aid in the design of
safer anticoagulant drugs.
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POSTER ABSTRACTS
POST 10-120
A Novel Activator of ATPase Activity of NBD1 Domain of the CFTR
Jay Singh, William Balch
Cell & Molecular Biology, The Scripps Research Institute, La Jolla, California, US
The most common Cystic Fibrosis disease is caused by the deletion mutation of phenylalanine at position
508 of CFTR, which is an ATPase super family protein. CFTR protein is made up of five domains; these
include two, six trans-membrane spanning domains, two nucleotide binding domains (NBDs) for ATP
bindings and hydrolysis, and a regulatory domain. To understand the mechanism of the ATPase activity of
NBDs of CFTR, we have developed a highly sensitive and very precise fluorescence based ATPase assay,
known as QR Assay. Using the principle that color product quenched the fluorescence signal of the white
microplates, by either absorbing excitation or emission signals. QR and phosphate ions (a product of ATP
hydrolysis) reaction give an intense red color in the solution that quenched the microplate fluorescence.
Interestingly, we found that acetate ions are capable to stimulate the intrinsic ATPase activity of NBD1
domain by through either dimerization or oligomerization of NBD1 domains. Acetate ions promote the
cross-linking of NBD1 monomer into dimmer or oligomer of human wild type NBD1 as well as deltaF508
NBD1, which is a sufficient and necessary condition for the enhancement of rate and extent of ATP
hydrolysis. Furthermore, we have collected evidence that light scattering of NBD1 domains are
substantially increased in the presence of acetate ions indicating the dimerization/oligomerization of
NBD1 domains. The associations between the NBD1 domains are ionic because the rate and extent of ATP
hydrolysis is decreased in the presence of various salts conditions. Our results strongly suggest that, ATP
hydrolysis control the CFTR channel activity through various combinations of salts and acetate ion in the
cells.
POST 10-121
Targeting the NEET Proteins for Cancer Treatment
Colin H. Lipper1, Mark L. Paddock1, José N. Onuchic2, Ron Mittler3, Rachel Nechushtai4, Emmanuel A.
Theodorakis1, Patricia A. Jennings1
1
Department of Chemistry and Biochemistry, University of California San Diego, San Diego, California,
US, 2Center for Theoretical Biological Physics and Department of Physics, Rice University, Houston,
Texas, US, 3Department of Biology, University of North Texas, Denton, Texas, US, 4Alexander Silberman
Institute of Life Science, Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem, Israel
The 2Fe-2S cluster NEET protein family members mitoNEET (mNT) and nutrient-deprivation autophagy
factor-1 (NAF-1) play key roles in the regulation of mitochondrial iron and reactive oxygen species
homeostasis. mNT localizes to the outer-mitochondrial membrane while NAF-1 localizes to the ER and
mitochondria-associated ER membranes. Recently, the NEETs were identified as potential targets for
cancer treatment. Both proteins were found to be overexpressed in human epithelial breast cancer cells,
and decreasing the level of either mNT or NAF-1 by shRNA knockdown to near normal levels significantly
reduced cancer cell proliferation and tumor growth and induced mitochondrial dysfunction in an iron
dependent manner [1]. The caged Garcinia xanthone derivative cluvenone localizes to the mitochondria
and induces apoptosis in cancer cells [2]. Cluvenone binds to both mNT and NAF-1, stabilizing the
oxidized 2Fe-2S cluster, and different cluvenone modifications can affect NEET cluster stability by either
stabilizing or destabilizing the cluster. We were the first to show that NEETs are cluster transfer proteins
[3] and our current findings indicate that the cluvenone compounds alter the transfer of the 2Fe-2S cluster
to apo-acceptor proteins. These results are consistent with the NEET proteins as cellular targets of the
cluvenone compounds with their effect on the FeS cluster responsible for their selective
cytotoxicity. References [1] Sohn YS, et al. (2013) Proc Natl Acad Sci USA 110(36):14676–14681. [2]
108
POSTER ABSTRACTS
Guizzunti G, et al. (2012) Invest New Drugs 30(5):1841-1848. [3] Zuris JA, et al. (2011) Proc Natl Acad Sci
USA 108(32):13047-13052.
POST 10-122
Understanding the Role of Human DDX21 RNA Helicase in HIV-1 Rev-RNA Assembly in vitro
Li Zhou, James R. Williamson
The Scripps Research Institute, La Jolla, California, US
The Rev protein of HIV-1 binds to the Rev Response Element (RRE) located in the env region of
incompletely spliced HIV mRNA and promotes nuclear export of intron containing viral transcripts. This
step is one of the hallmarks for the transition from early to late viral life cycle and viral infectivity. Several
host DEAD/H box RNA helicases are involved in this process. In particular, DDX21 silencing experiments
suggested that DDX21 is directly involved in the Rev-RRE dependent nuclear export in the late stage of
HIV infection, and it might play a role in the assembly of Rev-RRE complexes. To understand what role
DDX21 plays in Rev/RRE functions, we defined the direct interactions between DDX21 and Rev-RRE
complexes and explored the functional implications of these interactions. Using full length and truncated
recombinant DDX21 and Rev proteins purified from E. coli, we identified the interaction sites between
DDX21 and Rev via in vitro pull-down, limited proteolysis and fluorescence polarization assays. Our data
suggest that DDX21 associates with Rev in vitro, with the N-terminal DDX21 (the DExD domain) showing
similar effects, indicating a possible binding target. Moreover, the N-terminal 64 residues of Rev are
sufficient for interaction with DDX21. We also examined the effect of Rev-DDX21 binding on the
enzymatic properties of DDX21. Purified recombinant DDX21 displayed robust RNA-dependent ATPase
activity and ATP-dependent RNA unwinding activity. We first established the Michaelis-Menten
parameters for substrate ATP, and the apparent affinity for stem IIB RNA (high affinity binding site of Rev
on RRE RNA), and poly U RNA. We explored the effect of Rev on the RNA unwinding activity of DDX21 via
a real-time RNA unwinding assay using fluorescently labeled RNAs. Our results imply that the RNA
unwinding activity of DDX21 is not affected by the presence of Rev.
Poster Session: Proteins in Dynamic & Driven Processes
POST 11-123
Characterization of a Temperature Responsive Two-component Regulatory System in the Antarctic
Methanogen, Methanococcoides burtonii
Tahria Najnin1, Khawar S. Siddiqui1, Paul M. Curmi2, Ricardo Cavicchioli1
1
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington,
New South Wales, Australia, 2School of Physics, The University of New South Wales, Kensington, New
South Wales, Australia
Two-component regulatory systems (2CRSs) typically consist of a sensor histidine kinase and a response
regulator. Upon receiving a stimulus, the sensor histidine kinase autophosphorylates and transfers the
phosphoryl group to the receiver domain of a response regulator. The overall phosphorylation state is
governed by a combination of kinase and phosphatase activities mediated by the two proteins.
In Bacteria the response regulator tends to contain a helix-turn-helix (HTH) DNA binding output domain,
enabling the phosphorylated protein to bind DNA and regulate gene expression. In Archaea, only a
minority
of
response
regulators
contain
DNA
binding
domains.
The
Antarctic
109
POSTER ABSTRACTS
archaeon, Methanococcoides butonii, encodes numerous 2CRSs, and only a few response regulators
contain predicted HTH output domains [1]. The 2CRS consisting of Mbur_0694 (sensor histidine kinase)
and Mbur_0695 (response regulator with a DNA-binding output domain) is transcribed as an operon, and
the RNA and protein levels of Mbur_0695 are elevated during growth at low (4°C) vs high (23°C)
temperature [2, 3]. The upregulation at low temperature indicates this may represent a temperature
responsive 2CRS that is involved in regulating global gene expression in response to growth temperature
[3]. In order to characterize the structure, function and activity properties of this 2CRS, Mbur_0694 and
Mbur_0695 were cloned, overexpressed and proteins purified by affinity chromatography. The proteins
were characterized using size exclusion chromatography, liquid chromatography-mass spectrometry,
circular dichroism, differential scanning calorimetry and matrix-assisted laser desorption/ionization
techniques. In order to examine phosphate transfer during signal transduction the phosphorylation state
and phosphotransfer properties of the proteins were assessed using small phosphate donor molecules
including acetyl phosphate and carbamoyl phosphate. To facilitate studies of the response regulator, a
mutated version lacking the HTH domain was also examined. This presentation describes progress to
date, and represents the first report describing the biochemical properties of any 2CRS in Archaea.
POST 11-124
Crystal Structure of the Periplasmic Sensor Domain of Histidine Kinase CusS Bound to Silver
Trisiani Affandi1, Aaron V. Issaian1, Sue A. Roberts1, Megan M. McEvoy1, 2
1
Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, US, 2Soil, Water and Environmental
Sciences, University of Arizona, Tucson, Arizona, US
In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Each
two-component system consists of a sensor histidine kinase and response regulator, which work together
through histidyl-aspartyl phospho-relay. One of the two-component systems in Escherichia coli, CusSCusR, is known to induce expression of cusCFBA genes under increased periplasmic Cu(I) and Ag(I)
concentrations to help maintain metal homeostasis in cells. CusS is a membrane-associated histidine
kinase, such that the periplasmic sensor domain is connected with the cytoplasmic domain containing
ATP-binding and catalytic kinase domains through two transmembrane helices in the inner membrane.
However, the mechanism of how CusS senses increasing metal concentrations and activates CusR is not
yet known. Here, we present the crystal structure of the Ag(I)-bound periplasmic sensor domain of CusS at
a resolution of 2.15 Å. Two homodimers of the periplasmic sensor domain of CusS were found in the
asymmetric unit of a single crystal with four Ag(I) binding sites per homodimer. The crystal structure
reveals two symmetric metal binding sites at the dimeric interface between the two subunits that
coordinate Ag(I) through a unique site consisting of a His2 motif with a Ag(I)-π interaction between a
phenylalanine. Metal binding at the dimer interface is likely relevant to dimerization of periplasmic sensor
domain of CusS and activation of the cytoplasmic kinase core of CusS.
POST 11-125
Structural Investigation into the Mechanism of the Synthase Subunit of PLPS
Amber M. Smith1, 2, Janet L. Smith 1, 2
1
Biological Chemistry, University of Michigan, Ann Arbor, Michigan, US, 2Life Sciences Institute,
University of Michigan , Ann Arbor , Michigan, US
PLP synthase (PLPS) from the bacterium Geobacillus stearothermophilus generates pyridoxal 5'-phosphate
(PLP), the active form of vitamin B6, from glutamine, ribose 5-phosphate (R5P) and glyceraldehyde 3phosphate (G3P). PLPS is a D6-symmetric complex of 12 synthase subunits (PdxS) and 12 glutaminase
subunits (PdxT). Ammonia generated by glutamine hydrolysis in PdxT is channeled to the PdxS active site
where it joins R5P and G3P to form PLP by an unknown mechanism that proceeds through two sequential
110
POSTER ABSTRACTS
covalent intermediates in PdxS. First, R5P and PdxS(Lys81) form a Schiff base imine. Dehydration,
incorporation of NH3 from PdxT and loss of inorganic phosphate result in a chromophoric adduct of
1,2
unknown structure . Synthase activity is dependent on a PdxS C-terminal tail, which is disordered in all
structures reported to date. To investigate the mechanism of the synthase subunit and the role of the Cterminal tail of PdxS, we obtained three crystal structures of the synthase subunit alone (PdxS/R5P and
PdxS/R5P/NH3 complexes) or the intact enzyme (PLPS/R5P/Gln). The structures provide snapshots of PdxS
at distinct steps in its complicated catalytic cycle and provide insights into the elusive mechanism and
structural elements that drive the conversion of imine to chromophore. PdxS is highly dynamic,
alternating between open and closed conformations depending on the presence of PdxT and substrates.
The complex of PLPS with its substrates glutamine and R5P is the first structure of intact PLPS with
multiple substrates and an ordered C-terminal tail. The C-terminal tail interacts with the body of the
synthase subunit, acting as a shield over the R5P site in the closed state of the synthase subunit. The
structure of PdxS with R5P in the presence of NH3 captures three distinct states in the 12 active sites of
the crystallographic asymmetric unit, providing snapshots of PdxS from imine to chromophore formation.
Through biochemical assays and a comparison of the three crystal structures, we identified conserved
charged residues that stabilize the closed state that is essential to formation of the chromophore.
Supported by NIH grant DK42303. 1.Raschle, T. et al. The Journal of biological chemistry 282, 6098-105
(2007). 2. Hanes, J.W. et al. Journal of the American Chemical Society 130, 3043-52 (2008).
POST 11-126
Super Spy Variants Implicate Flexibility In Chaperone Action
Shu Quan2, 1, Lili Wang2, 3, Evgeniy V. Petrotchenko4, Karl A. Makepeace4, Scott Horowitz2, 3, Jianyi Yang5,
Yang Zhang5, Christoph H. Borchers4, James C. Bardwell2, 3
1
State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of
Science and Technology, Shanghai, China, 2Molecular, Cellular, and Developmental Biology, University of
Michigan, Ann Arbor, Michigan, US, 3Howard Hughes Medical Institute, Chevy Chase, Maryland,
US, 4Department of Biochemistry and Microbiology-Genome British Columbia Proteomics Centre,
University of Victoria , Victoria, China, 5Department of Computational Medicine & Bioinformatics,
University of Michigan, Ann Arbor, Michigan, US
Experimental study of the role of disorder in protein function is challenging. It has been proposed that
proteins utilize disordered regions in the adaptive recognition of their various binding partners. However
apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has
proven to be difficult. Disorder is an emerging theme, not only among many hub proteins but also among
molecular chaperones, which in general allow promiscuous substrate binding. We have utilized a genetic
selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered
chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates.
Based on our biochemical and biophysical evaluation of these "Super Spy" variants, there appear to be
multiple factors that result in the increased chaperone activity. In addition to increasing binding site
hydrophobicity, most of our super-Spy variants show tighter binding to client proteins and are generally
more unstable than is wild type Spy. These variants also show increased levels of disorder in the absence
of client proteins, indicating increases in apparent flexibility of these variants. We establish a good
relationship between the degree of their instability and the improvement they show in their chaperone
activity. Our results provide evidence for the importance of disorder and apparent flexibility in chaperone
action.
POST 11-127
Elucidation Of The Nonspecific DNA-Binding Mechanism Of The POU Homeodomain Using NMR
111
POSTER ABSTRACTS
Tsuyoshi Konuma1, Erisa Harada1, Takashi Oda2, Mamoru Sato2, Kenji Sugase1
1
Bioorganic Research Institute, SUNTORY foundation for life sciences, Osaka, Japan, 2Graduate school of
medical life science, Yokohama city university, Yokohama, Kanagawa, Japan
Transcription factors locate effectively to their target DNA. It is generally accepted that a protein first
binds nonspecifically to DNA, and then searches for its specific site. However, the details of the dynamic
search process are still largely unknown. In this study, we have investigated dynamics of POU
homeodomain (POUHD) of Oct4 upon DNA binding using NMR. Firstly, we titrated 16-bp DNA into
POUHD to characterize the conformational changes and binding kinetics upon DNA binding. Interestingly,
almost all peaks in HSQC spectra shifted sigmoidally. They could not be fitted to the simple two-state
binding model; therefore, nonspecific bound states should be taken into account to explain the titration
data. Here, we constructed a new binding model including the nonspecific bound states using the BlochMcConnell equation, which describes the time evolution of magnetization including the chemical
exchange effect. The fitting of the chemical shift changes to the new model yielded kinetic parameters for
POUHD binding to DNA. Subsequently, to obtain structural information on the nonspecific bound states,
CLEANEX-PM, which determines exchange rates between amide and water protons, was applied to
POUHD in the presence of excess amounts of POUHD over DNA to increase the population of the
nonspecific bound states. As a result, the amide protons of the N-terminal loop are protected from
solvent by the bound DNA in the nonspecific binding states, whereas those of the α3-helix remain to be
exchangeable with water protons. Therefore, it is suggested that the N-terminal loop first interacts with
nonspecific sites of DNA, and then the stable complex with DNA is formed by locating the α3-helix to its
specific target site. This mechanism corresponds to the fly-casting model.
POST 11-128
Time-lapsing Planaria: Studying Planarian Regeneration by Stop-motion Imaging and by Pulseisotopic Proteome Labeling
Wei Shen1, Karsten Berning2
1
Department of Orthopaedics and Traumatology, The University of Hong Kong 2Department of Biology
and Chemistry, City University of Hong Kong, Hong Kong
The unraveled regenerative capacity of planarian makes it a valuable model organism for studying the
molecular and cellular basis of tissue repair and regeneration. The combined approach of using time-lapse
microscopy and quantitative proteomics has revolutionized modern biology by enabling the
characterization of the dynamics of complex biological processes, but is seldom applied to planarian
research. One main reason is the strong photophobicity of planarians that makes the imaging of living
worms extremely difficult. Many attempts have been made on paralyzing planarians for imaging.
However, these methods are tedious and relatively ineffective. Here, we report a new method that
involves the use of agarose gel to embed the planarian Dugesia Japonica of typical size (2–6 mm),
rendering them immobilized for as long as four days without the use of any chemical or genetic
manipulation. Using this method, we documented the first time-lapse movie of the whole regeneration
process of an amputated worm. In addition, we incorporated various fluorescent dyes in our imaging,
allowing the capture of the single cell movements in planarian epidermis. In parallel, we used quantitative
proteomics to characterize the biochemical changes during planarian regeneration during the same time
window. Like many other proteomic approaches, the presence of large pools of stable proteins in the
planarian proteome often masks the fluctuations of regulatory factors, which are often present in low
abundance. To circumvent this obstacle, we fed unperturbed or regenerating planaria with SILAC-encoded
human cells, and observed the assimilation of the isotopically labeled amino acids from the human cells
into the planarian proteome over the course of four days. We envisage that the rate of assimilation
reflects the flux of protein biosynthesis activity required during regeneration. Using this technique, we
112
POSTER ABSTRACTS
identified calponin-2 as a highly-synthesized proteins in head-regenerating, but not in tail-regenerating,
planaria, thus confirming the importance of calcium homeostasis in the maintenance of planarian body
plan. Taken together, the two techniques developed in this work reveal previously unknown aspects of
planarian regeneration, and can potentially extend our understanding of the biology of this fascinating
organism.
POST 11-129
Hydrogen Deuterium Exchange Mass Spectrometry and Molecular Dynamics Reveal the
Interactions of Membrane Phospholipids and Inhibitors with Phospholipases A2
Varnavas Mouchlis, Denis Bucher, J. Andrew McCammon, Edward A. Dennis
UC San Diego, La Jolla, California, US
Phospholipase A2 (PLA2) constitutes a superfamily of enzymes which catalyze hydrolysis of the ester bond
at the sn-2 position of membrane phospholipids (Chem. Rev. 2011, 111, 6130). The products of
PLA2 activity are free fatty acids, predominantly arachidonic acid (AA), and lysophospholipids. The AA is
further metabolized by COX-1, COX-2 and 5-LO to form a variety of pro-inflammatory lipid mediators
including prostaglandins and leukotrienes. Among the members of the PLA2superfamily, GVIA iPLA2 and
GIVA cPLA2 have a similar size and they function through a Ser/Asp catalytic dyad located in an α/βhydrolase domain. However, GIVA cPLA2 shows specificity for phospholipids containing arachidonic acid,
while the GVIA iPLA2 shows low specificity. These enzymes have been associated with several
inflammatory diseases and thus they are attractive targets for inhibitor development. Both enzymes have
been expressed using the Baculovirus expression system. Group-specific assays were developed for
testing the inhibitory activity of small compounds against both enzymes. Deuterium exchange mass
spectrometry (DXMS) and computer-aided drug design were employed to study the binding interactions
of PAPC and inhibitors. DXMS revealed important information about regions of the enzymes interacting
with the membrane and inhibitors. The DXMS information and Induced Fit docking have been used to
predict the binding mode of PAPC and inhibitors, and to create complexes of the enzymes with a
modeled membrane (PLoS Comput. Biol. 2013, 9, 1 and J. Am. Chem. Soc. 2013, 135, 1330). The
substrate- and ligand-enzyme complexes revealed key residues that play important roles in binding.
Further studies of the complexes with molecular dynamics (MD) simulations have been employed to
interpret the DXMS results (J. Biol. Chem. 2013, 288, 1806) and determine the conformational changes
resulting from the binding process. Long MD simulations with the Anton supercomputer revealed two
states for the enzyme, “closed” and “open”, suggesting that the membrane acts as an allosteric ligand
shifting the enzyme to the “open” state. This study provides insight into the interactions of the enzyme
with membrane, substrate and inhibitors, and this information will be useful in developing new inhibitors
with improved properties. (Supported by NIH RO1 GM 20,501-38; Anton was provided by the National
Center for Multiscale Modeling of Biological Systems).
POST 11-130
Visualizing the Inter-domain Motions of a Flexible Protein Using Continuous Models
Yang Qi1, Jeffrey W. Martin2, Bruce R. Donald2, 1, Terrence G. Oas1
1
Biochemistry, Duke University, Durham, North Carolina, US, 2Computer Science, Duke University,
Durham, North Carolina, US
SpA-N is the N-terminal half of Staphylococcal protein A and it is composed of five protein binding
domains. The five domains could bind to antibody, TNFR1 and von Willebrand factor and facilitate the
evasion of Staphylococcus aureus into the human immune system. The functional plasticity could result
from structural flexibility. We first observed motions of SpA-N in sub-nanosecond timescale using
heteronuclear NMR relaxation. The result suggests that the five domains of SpA-N are connected by four
113
POSTER ABSTRACTS
flexible linkers. In order to observe dynamics in a wider range of timescales, we constructed a di-domain
mimic of SpA-N with a lanthanide binding tag (LBT) and measured residual dipolar couplings (RDCs). The
di-domain construct can bind lanthanide ions and consequently be aligned in the presence of a strong
magnetic field. By using different lanthanide ions, we also obtained multiple alignments, which contain
additional dynamic information than a single alignment. In addition, we designed a de novo method to
extract dynamic information from RDCs. Instead of determining conventional structure ensembles, our
method determines an inter-domain orientation distribution to describe the structure of a flexible protein
using continuous probability distributions. The method can avoid the over-fitting problem and generate a
least biased model. Using the method, we determined the inter-domain orientation distribution of the didomain construct. A strong correlation was observed in the distribution and conformations were well
populated in a limited region. Based on the result, we hypothesized that protein A is able to reach
important biological conformations with a high probability and avoid other unnecessary conformations. If
so, the conformation space of protein A makes protein A a versatile virulent protein by minimizing the
entropy cost to bind to a binding partner.
POST 11-131
Benchmarking FRET for Live-cell PPI
Tao Lin1, Brandon Scott1, Francisca Essel1, Moul Dey2, Adam Hoppe1, Suvobrata Chakravarty1
1
Chemistry & Biochemistry, South Dakota State University, Brookings, South Dakota, US, 2Health &
Nutritional Sciences, South Dakota State University, Brookings, South Dakota, US
In recent years, fluorescence resonance energy transfer (FRET) has become a powerful tool to study
molecular interactions, especially protein-protein interactions (PPI). Recombinant fluorescent proteins
enable the study of such interaction in living cells. Whilst the dynamics of live cells are known as the
ultimate analysis, the limits of the strength of molecular interactions that can be detected by live-cell FRET
remain poorly understood as no appropriate indicator is available for this. To meet the demand on FRET
reporter molecules for live-cell, we introduce a new facile sensor analysis strategy. Using a test set of 11
pairs of small interacting proteins for whom the respective affinities range between mM – nM, we
determine protein concentration in living cells to compute the interaction affinity using the intra-cellular
FRET efficiency. Furthermore, we also compare affinity obtained from FRET with that obtained from ITC. All
of these results suggest that we establish the limits with live-cell microscopy. This study benchmarks
intracellular FRET and is a very useful reference for intracellular PPI methods (BiFC, IDPRIME etc).
POST 11-132
Dynamics And Chaperone Function In The Small Heat-Shock Protein αB-Crystallin
Georg Hochberg1, Heath Ecroyd2, Dezerea Cox2, Michael Sawaya3, Cong Liu3, Duilio Cascio3, Miranda
Collier1, James Stroud3, John Carver4, Andrew Baldwin1, Carol Robinson1, David Eisenberg3, Justin
Benesch1, Arthur Laganowsky1
1
Chemistry Research Laboratory, Oxford University, Oxford, United Kingdom, 2University of Wollongong,
Wollongong, New South Wales, Australia, 3University of California, Los Angeles, Los Angeles, California,
US, 4The Australian National University, Canberra, Australian Capital Territory, Australia
Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and
dynamic complexes with target proteins, preventing their aggregation into either amorphous deposits or
amyloid fibrils. How sHSPs carry out their important function is unknown, but it is generally believed to
depend on their complex quaternary dynamics, including the formation of large and heterogeneous
oligomers, their inter-conversion via subunit exchange, and the presence of disordered terminal domains.
Although these dynamics can now be accurately measured using native mass spectrometry and nuclear
magnetic resonance (1), the heterogeneity inherent in this system makes it difficult to test conclusively
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POSTER ABSTRACTS
which aspects of sHSP assemblies are required for chaperone function. To overcome these challenges, we
engineered truncated constructs of the two most abundant sHSPs in human tissue, αB-crystallin and
HSP27 in a manner allowing us to carefully control their quaternary dynamics and solve their structures by
X-ray crystallography (2). We quantified the quaternary dynamics of these domains using native mass
spectrometry, and used engineered cysteines to drive their equilibrium stoichiometries from rapidly
interconverting monomers and dimers to conformationally restricted dimers that cannot exchange
subunits.
Remarkably, we find that the αB-crystallin core domain alone has chaperone activity
comparable to that of the full-length protein, despite its inability to form large oligomers and lack of
disordered terminal domains and regardless of whether the αB-crystallin core domain is locked into a
dimer or predominantly monomeric. Furthermore, it is a potent inhibitor of amyloid fibril formation and,
by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Our
experiments therefore identify a novel, small and highly structured ‘functional unit’ of the heterogeneous
sHSP
oligomeric
ensemble,
potentially
enabling
more
rational
design
of
amyloid
inhibitors. 1.
Hochberg G & Benesch J (2014) Dynamical structure of αB-crystallin. Prog. Biophys.
Mol. Biol. doi: 10.1016/j.pbiomolbio.2014.03.003. 2.
Hochberg G, et al. (2014) The structured core
domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity. Proc. Natl. Acad. Sci.
USA 111(16):E1562-E1570.
POST 11-133
Protein Flexibility and Gymnastics Drive Robust Clockwise Ticking of a three-protein KaiABC
Oscillator
Yonggang Chang, Roger Tseng, Andy LiWang
University of California, Merced, Atwater, California, US
Almost all living organisms have evolved endogenous circadian (~24 h) clocks in anticipation of daily
environmental changes. The discovery of the cyanobacterial clock has made feasible mechanistic
understanding of circadian clocks in that (1) its circadian oscillator is simple, composed of only three
proteins KaiA, KaiB, and KaiC; and (2) the oscillator can be reconstituted in a test tube. Our goal is to
eventually understand how the three proteins work together to generate sustained rhythms. KaiC
displays a 24-h rhythm of phosphorylation under the positive regulator KaiA, which promotes KaiC autophosphorylation, and the negative regulator KaiB, which promotes KaiC auto-dephosphorylation. KaiC is a
homohexamer and each subunit has two homologous domains, each of which associates into rings,
termed CI and CII, respectively. KaiC has two neighboring phosphorylation sites Ser431 and Thr432 and its
phosphorylation cycle occurs in an ordered pattern ST->SpT->pSpT->pST->ST, where S and T stand for
S431 and Thr432, respectively, and pS and pT mean that the two sites are phosphorylated. This ordered
phosphorylation has remained unclear. Here we show that the phosphorylation on the two sites has
distinct effects on the CII ring flexibility, with the Thr432 phosphorylation maintaining a flexible CII ring
and the Ser431 phosphorylation rigidifies the CII ring. Upon Ser431 phosphorylation-induced CII ring
rigidification, the CII ring stacks with the CI ring, which triggers the exposure of the KaiB-binding site on
the CI ring for KaiB to recruit KaiA away from the CII ring to the CI ring, initiating KaiC autodephosphorylation. We conclude that protein flexibility underpins the clockwise directionality of the
oscillator. Finally, we show that the robustness of the oscillator hinges on the slow (hours timescale) KaiBKaiC binding, the critical delay for KaiA inhibition. We find that the exceptionally slow binding is in large
part due to it that KaiB undergoes a global fold change. Such global fold change could be more widely in
other timing-related events such as cell cycle.
POST 11-134
Characterization Of Dynamic Processes In Substrate Recognition By Cytochrome P450 Enzymes
115
POSTER ABSTRACTS
Nitin Jain, Nicholas Lopes, Ana Bernal
University of Tennessee, Knoxville, Tennessee, US
Apart from structural factors, conformational flexibility of cytochrome P450 enzymes plays a key role in
modulating protein-substrate interactions to accommodate substrates of differing physico-chemical
properties as well as product specificity. Conformational dynamics of Cytochrome P450cam (CYP101) in
various substrate-bound forms have been investigated using a combination of solution NMR spectrocopy
and MD simulations to identify key regions undergoing dynamic motions and quantitate the flexibility.
Differential dynamics are observed on all time-scales in several regions of CYP101 in presence of various
substrates, indicating dynamic selection of CYP101 conformational space by different substrates. Similar
conformational dynamic effects have been observed in the complex between CYP101 and its redox
binding partner putidaredoxin (Pdx) in terms of substrate turnover. We present the data from these
dynamic studies which clearly validate the role of dynamic processes in substrate recognition by CYP101.
POST 11-135
Mapping the Interactions Between the Molecular Chaperones Hsp70, Hsp104 and Hsp110
Shankar Shastry, Shannon Doyle, Joel Hoskins, Sue Wickner
National Cancer Institute, Bethesda, Maryland, US
Molecular chaperones help maintain cellular protein quality by assisting in the proper folding of nascent
and misfolded proteins, preventing protein aggregation, reactivating aggregated proteins and targeting
unfolded and misfolded proteins for degradation. Protein aggregation is linked to many diseases
including Alzheimer’s disease, Parkinson’s disease, amyloidosis and cancer. In E. coli, the ClpB chaperone
collaborates with DnaK and its cochaperones, DnaJ (an Hsp40) and GrpE (a nucleotide exchange factor) to
disaggregate and reactivate aggregated proteins. In yeast, Hsp104 disaggregates proteins in conjunction
with Ssa1, a yeast Hsp70, and its cochaperones, Ydj1 or Sis1 (Hsp40s) and Sse1 (a nucleotide exchange
factor). This collaboration between chaperones is specific: E. coli DnaK only functions with E. coli ClpB and
yeast Ssa1 acts with yeast Hsp104. We identified regions on E. coli DnaK important for collaboration with
ClpB. These residues reside in a portion of the GrpE binding site on DnaK, as previously observed for the
interaction between DnaK and ClpB from Thermus thermophilus (Rozenzweig et al, 2013, Science). Since
the interactions between chaperones are specific, we wanted to determine if the residues on Ssa1 that
interact with Hsp104 are in the same region as on DnaK. Mutations were generated in Ssa1 and the
proteins were purified. When we tested for the ability of the mutants to reactivate inactive protein
aggregates with Hsp40 in the presence or absence of Hsp104, we identified Ssa1 mutants that showed
defects in collaboration with Hsp104. These mutations were in residues homologous to residues identified
as being important for the interaction between DnaK and ClpB. Based on the crystal structure of bovine
Hsc70 in complex with Sse1 (Schuerman et al, 2008, Mol. Cell), the residues in Ssa1 that interact with
Hsp104 would also be expected to interact with Sse1. [NN2] By uncovering the specifics of the
interactions of Hsp70 with Hsp104 and Hsp110, we are able to describe a more comprehensive model for
protein disaggregation.
POST 11-136
The Role Of Phenylalanine In An Intrinsically Disordered Protein From Yeast Nucleoporins
Korey M. Reid1, Krish Krishnan1, 2
1
Chemistry, California State University Fresno, Fresno, California, US, 2Department of Pathology and
Laboratory Medicine, University Of California, Davis, Sacramento, California, US
Intrinsically disordered proteins (IDP) are a class of proteins that do not adopt a well defined threedimensional structure, and are characterized by their minimally folded structure with high intramolecular
flexibility under physiological conditions. Their structural disorder directly or indirectly, plays an important
116
POSTER ABSTRACTS
role in many essential cellular and regulatory processes. The nuclear pore complex (NPC) is a large protein
complex that is the sole gateway for transport of proteins and RNAs between the nucleus and cytoplasm
of a cell. It is composed of approximately 30 nucleoporins, or nups, a third of which are intrinsically
disordered and contain a high count of FG-domain repeats (FG-nups). These FG-nups occupy the central
pore region of the NPC and play an integral role in the regulation of nucleocytoplasmic transport of
macromolecules through the NPC. It is hypothesized that the FG-Nups function in the NPC by forming
either cohesive elements mediated through –GLFG- rich motifs or non-cohesive elements mediated
through –FG- or –FxFG- motifs. In this study we focus on understanding the role of Phe (F) residues in the
structural ensemble formed in a model FG-nup peptide. A 25 AA model peptide (FGnup) is designed
using the native sequences of yeast nups containing three FG-domains, two of which are GLFG-motifs. To
understand the role of Phe, a variant peptide (AGnup), with a substitution of Phe with Ala is also
considered. Molecular conformations of these peptides are determined using modern nuclear magnetic
resonance (NMR) methods and all atom molecular dynamics (MD) simulations. Multi-dimensional NMR
experiments combined with distance geometry algorithms are used to determine the ensemble of
conformations of the two peptides. All atom MD simulations were performed with multiple solvent
models to understand residue specific details of intra-molecular interactions. Experimental results suggest
that the FGnup is slightly more compact than the AGnup. However, MD results suggest that the average
radius of gyration of the FGnup is greater than that of the AGnup. One possible reason for the difference
between the NMR and MD simulations could be the time-scale of the dynamics sampled between these
two methods. Detailed analysis of the ensemble of conformations obtained experimentally and using
simulations will be presented, in addition to the implications of these results on the role of intrinsic
disorder in the function of FG-nups.
POST 11-137
The Acidic Residues of the IκBα PEST Sequence are Responsible for Actively Dissociating NFκB from
DNA
Holly E. Dembinski, Kevin Wismer, Elizabeth Komives
Chemistry and Biochemistry, UCSD, La Jolla, California, US
Nuclear factor kappa B (NFκB) transcription factors are responsible for the regulation of hundreds of
target genes, their expression is induced by many classes of stimuli, and NFκBs play essential roles in the
healthy regulation of cellular development and proliferation in inflammatory and immune
responses. Diseases such as cancer, heart disease, Alzheimer’s disease, and AIDS can be attributed to the
aberrant regulation of NFκB. The transcriptional activity of NFκB is controlled by its inhibitors, the
IκBs. IκBα, in particular, dynamically responds to extracellular stimuli releasing a burst of NFκB that enters
the nucleus and activates target genes. The transcriptional activation is short-lived, and our lab has been
investigating the mechanism of post-induction repression. We previously showed that IκBα actively
dissociates NFκB from DNA. Analysis of the crystal structures of NFκB (RelA/p50) with DNA and with IκBα
shows that the IκBα PEST sequence, which is rich in glutamate and aspartate residues, forms similar
electrostatic contacts to NFκB as the DNA. Given this, we hypothesized that the IκBα PEST sequence
electrostatically repels DNA from NFκB during the dissociation process. Here we present fascinating
results that show that the individual and collective, conservative mutation of these acidic residues to their
amide counterparts does not affect the binding affinities of these mutants to NFκB; however, the mutant
in which all five acidic residues are neutralized is incapable of actively dissociating NFκB from DNA and
intriguingly forms a stable IκBα-NFκB-DNA ternary complex. We are currently investigating the structure
of this ternary complex via nuclear magnetic resonance, and we are exploring the effects of this mutation
and the importance of the accelerated dissociation of NFκB from DNA by IκBα in mouse embryonic
fibroblast cells.
117
POSTER ABSTRACTS
POST 11-138
Nanoscale Hydrodynamic Study of Proteins under Thermal Agitation and Electric Field
Yuanming Zhang, Zachary Weiner, Eric Farrell
Brookhaven Instruments Corporation, Holtsville, New York, US
Proteins hydrodynamics is characterized at nanometer scale with the temporal analysis of their Rayleigh
scattering. The dynamic-structure-factor is experimentally extracted and used to quantify proteins meansquare-displacement under thermal agitation and electrophoretic motion under electric field. Through the
subsequent data analysis based on molecular theories of proteins hydrodynamics, it is demonstrated that
valuable insights can be obtained regard the physical characteristics of proteins.
Hierarchic structures and multiscale dynamics of proteins give rise to their versatile biological
functionalities that play vital roles in DNA transcription, mRNA translation, signal transduction, and etc..
These complex biological processes are enacted or controlled through protein transport and interactions.
The subject receives increasing attentions not only for understanding of bio-molecular and cellular
functions, but also for pharmaceutical development. In order to analyze protein dynamics in a complex
biological process, proteins often need to be selectively probed either by immobilization or by labeling.
Due to unwanted dynamic alternation and measurement bias caused by immobilization or labeling, labelfree methods are therefore optimal to probe protein dynamics in their native biological environment.
Ubiquitous Rayleigh scattering is a viable tool for the purposes of in-situ and label-free probe on
proteins hydrodynamics at broad length scale. Protein hydrodynamics can be readily quantified with
spectral broadening and shift of the light signal scattered from them. In the present study, using common
proteins such as lysozyme and Bovine Serum Albumin (BSA), we demonstrate the sensitivity and precision
of the techniques. The hydrodynamic and other physical characteristics of proteins, such as translational
diffusion coefficient, charge and electrostatic force/potential range (Debye length), can then be calculated
with Stokes-Einstein equation of translational friction force and Debye-Hückel-Henry theory of
electrophoretic effect. Furthermore, it is shown that the effects of solvation/suspension media as well as
protein-protein interaction can be assessed well within the measurement certainty.
POST 11-139
Probing the Clamping Movement of Xylanase B by NMR Spectroscopy
1, 2, 3
1, 2, 3
Nhung T. Nguyen
, Nicolas Doucet
1
INRS-Institut Armand-Frappier - University of Quebec, 531 boul. des Prairies, , Laval, H7V 1B7, Quebec,
Canada, 2PROTEO, the Québec Network for Research on Protein Function, Structure, and Engineering,
1045 Avenue de la Médecine, Université Laval, Quebec, G1V 0A6, Quebec, Canada, 3GRASP, the Groupe
de Recherche Axé sur la Structure des Protéines, 3649 Promenade Sir William Osler, McGill University,
Montreal, Quebec, Canada
The present work describes NMR assignments and relaxation dispersion experiments probing the
dynamics of apo and ligand-bound xylanase B (XlnB) from Streptomyces lividans. Evidence from
mutagenesis, crystal structures and molecular dynamics simulations previously suggested that the
15
“thumb-loop” motion of XlnB might play a major role in substrate binding/catalysis [1,2,3,4,5]. Our NCPMG data show similar millisecond time-scale motions for active-site fingers in the free and xylobiosebound enzyme. However, in the presence of the longer xylopentaose ligand, conformational exchange
1
15
emerges on the thumb loop, along the active-site cleft, and on the opposite side of the fingers. H- N
HSQC titration data also indicates the involvement of thumb loop and binding cleft residues in substrate
recognition. For the first time, our results illuminate the atomic-scale dynamics of XlnB on the millisecond
time-scale, suggesting a global clamping movement during catalysis.References: [1]. Paës G., Berrin J.G.,
and Beaugrand J. Biotechnology Advances. (2012) 30: 564–592. [2] Pollet A., Lagaert S., Eneyskaya E.,
118
POSTER ABSTRACTS
Kulminskaya A., Delcour J.A., and Courtin C.M. Biochimica et Biophysica Acta (2010) 1804:977–985. [3].
Murakami M.T., Arni R.K., Vieira D.S., Degrève L., Ruller R., and Ward R. FEBS Letter (2005), 579:6505–10.
[4]. Vieira D.S, Degreve L., and Ward R.J. Biochimica et Biophysica Acta-Gen Subject (2009); 1790:1301–6.
[5]. Hakulinen N., Turunen O., Janne Janis J., Leisola M. and Rouvinen J. European Journal of
Biochemistry (2003), 270, 1399–1412.
POST 11-140
Intrinsic GTP Hydrolysis is Observed For a Switch 1 Mutant of Cdc42 in the Presence of a Specific
GTPase Inhibitor
Reena Chandrashekar, Kyla Morris, Colin D. Heyes, Paul D. Adams
Chemistry and Biochemistry, Univeristy of Arkansas-Fayettevillle, Fayetteville, Arkansas, US
The Ras-related protein Cell division cycle 42 (Cdc42) is important in regulating cell-signaling processes.
Cdc42-protein interactions are targeted towards flexible “Switch” regions in the Ras protein that help
regulate effector binding. We have studied the kinetics of the intrinsic GTP hydrolysis reaction in the
absence and presence of a peptide derivative of a p21-activated kinase effector (PBD46) for a Switch 1
mutant of Cdc42, Cdc42(T35A), and compared it to that of Cdc42 wild type. While the mutation in Cdc42
does not affect GTP hydrolysis, the binding between Cdc42 and PBD46 is disturbed. Moreover, whereas
the binding of PBD46 to wild type Cdc42 results in complete inhibition of GTP hydrolysis, the same
interaction in Cdc42(T35A) leads to a partial recovery of GTP hydrolysis. The kinetics, together with the
concentration dependence of PBD46 on the degree of inhibition of GTP hydrolysis suggest that the
dynamics changes in the Switch 1 region of Cdc42 caused by the T35A mutation fosters very slow
interconverting conformational states. We rationalize a model where both of these states possess the
ability to hydrolyze GTP, but one that has a drastic difference in its ability to interact with PBD46. Our
findings suggest that one way to control potentially oncogenic Ras protein over-activity may be facilitated
by altering conformational dynamics underlying Ras-protein interactions that can lead to abnormal Rasstimulated cell signaling.
POST 11-141
Three-dimensional Structure of the 54 kDa Subunit of the Chloroplast Signal Recognition Particle
using Molecular Modeling
Rory Henderson1, Suresh Kumar1, Colin Heyes1, Ralph Henry2
1
Chemistry & Biochemistry, University of Arkansas, Fayetteville, Arkansas, US, 2Biological Sciences,
University of Arkansas, Fayetteville, Arkansas, US
The chloroplast signal recognition particle is a heterodimeric complex of the 54kDa cytosolic signal
recognition particle homologue, cpSRP54, and a novel 43kDa protein, cpSRP43. While a nearly complete
structure for cpSRP43 has been obtained via X-ray crystallography, no structure is yet available for
cpsRP54. A structure for this protein could provide valuable information for the rationalization of the
extensive information already available regarding its function, and in the understanding of the as yet
undetermined mechanism of light harvesting chlorophyll binding protein’s (LHCP) insertion into the
thylakoid membrane. In this investigation we developed a structure for cpsRP54 using a combination of
homology modeling, de novo structure prediction and molecular dynamics simulation. The resulting
structure is consistent with the known properties of the protein and sheds light on some of the
mechanistic details of its functioning.
POST 11-142
Evidence For Significant Changes In Backbone Motions Between Apo And Gα-Bound Human RGS4
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POSTER ABSTRACTS
Lusine Simonyan, Shayla A. Brooks, Karin A. Crowhurst
Chemistry and Biochemistry, California State University Northridge, Northridge, California, US
RGS proteins act as essential regulators of G protein activity by accelerating GTP hydrolysis by Gα
subunits, terminating signaling and ultimately controlling the relay of various neurological signals from
outside to inside cells; they therefore play a central role in signal transmission, neuronal development and
synaptic plasticity. It is now understood that protein motions, flexibility and conformational changes often
have key roles in signal transmission processes. Numerous studies have been performed on the RGS and
Gα domains as potential drug targets; consequently, crystal and NMR structures of RGS proteins have
been solved. However, despite reports of preliminary evidence for protein flexibility and conformational
changes in Gα-RGS interactions, they have not been characterized in detail. One of our project goals has
been to compare the internal dynamics of apo versus Gαi1-bound human RGS4 (hRGS4) in order to
improve our understanding of the regulation of signal transmission. In the long term we would like to
compare these results with data from hRGS7 (which also binds Gαi1 but with lower affinity) in order to gain
insight into the role of dynamics on the selectivity of RGS proteins for their Gα targets. In our current
phase of the project we have used NMR spectroscopy to record fast and intermediate timescale backbone
relaxation as well as hydrogen exchange experiments on apo hRGS4. Results indicate that the N- and Ctermini are the primary sites of increased fast (ps-ns) timescale motions; interestingly, intermediate (μsms) timescale motions were observed on the helix of the primary binding site with Gα. Unexpectedly, we
also discovered that hRGS4 maintains a dimer structure, even at low concentrations, whose interface must
still be characterized. We have also recorded preliminary experiments on isotopically labeled hRGS4
bound to unlabeled (i.e. NMR invisible) Gαi1. An overlay of HSQC NMR spectra from apo and Gαi1-bound
hRGS4 confirm evidence (from NMR and crystal structures) for a conformational change when hRGS4
binds its target. However, it was a surprise to see how extensive the chemical shift changes were: almost
every backbone resonance was shifted to some extent. Further investigation of these changes should
provide increased insight into the role of protein motions in RGS-Gα signaling interactions. We gratefully
acknowledge support from NSF grants MCB-1158177 (RUI) and CHE-1040134 (MRI).
Poster Session: Protein Engineering & Synthetic Biology
POST 12-143
Engineering Picomolar Affinity into a Rationally Identified 5 kDa Scaffold for Tumor Targeting
Max Kruziki, Patrick Holec, Benjamin Hackel
Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, US
Small protein scaffolds, relative to antibodies, can provide superior stability, production, conjugation, and
distribution. Yet while many scaffolds have been tested to generate synthetic ligands, the causes of the
varying degrees of success or failure have not been systematically explored. We merged computational
and experimental tools to elucidate the biophysical factors that dictate evolutionary potential to empower
rational identification or design of protein scaffolds. We have identified the 45-amino acid Gp2 domain as
an effective protein scaffold to engineer stable, picomolar affinity ligands.
We developed and
implemented a computational algorithm to efficiently evaluate naturally occurring protein domains on
numerous metrics with potential impact on evolution of binding function. In one implementation, we
identified all domains in the Protein Data Bank with two accessible loops (diversified paratope) and scored
them on the size, shape, orientation, and target accessibility of their paratopes as well as protein size,
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POSTER ABSTRACTS
stability upon mutation, and dependence on disulfide bonds and/or cofactors. Machine learning was used
to preliminarily weigh each metric based on historical scaffold performance. The top scaffold was the 45residue T7 phage gene protein 2 with a single alpha helix opposite a beta sheet with two adjacent loops
amenable to mutation. We diversified the twelve loop amino acids using complementarity bias and length
8
diversity. The library of 10 mutants was displayed on the yeast surface and screened for binders to
several protein targets. Novel ligand discovery and directed evolution yielded binders with affinities as
strong as 200±100 pM and no observable nonspecific binding. Circular dichroism showed secondary
structure comparable to the wild-type protein and Tm values from 70±4ºC to 80±1ºC for evolved ligands
relative to 67±4º for wild-type. We evolved binders to epidermal growth factor receptor that effectively
label human tumor cells with nanomolar affinity. More broadly, libraries from the top 30 scaffolds were
created for comparative analysis of the ability to evolve specific, high affinity binding to numerous targets.
We will discuss the relative efficacy of each scaffold as well as the scaffold design metrics that correlate
most strongly with performance. Implications for future scaffold design, including synthetic protein
topologies, will be addressed as well as other designs.
POST 12-144
Utilizing Metal-Ligand Interactions to Promote Assembly of Collagen-Based Peptides into
Functional Nanostructures
Jeremy Gleaton, David Przybyla, Charles M. Rubert-Perez, Jean Chmielewski
Purdue University, Lafayette, Indiana, US
Collagen is a highly abundant protein that comprises up to one third of the total protein content within
humans. Collagen serves as an essential scaffold and structural support throughout nature. Because of
collagen’s striking structural strength and importance, efficient methods for mimicking natural collagen
and the development of collagen biomaterials are actively being pursued. Here we present a collagen
mimetic peptide, modified with bipyridines, that undergoes a hierarchical assembly into triple helices,
followed by the formation of curved disks via hydrophobic interactions. Metal-ion mediated assembly of
the curved disks is demonstrated to form hollow microspheres. These hollow microspheres encapsulate
various molecular weight fluorescently labeled dextrans, and demonstrate a time dependent release of
contents under a variety of thermal conditions. By gaining an understanding of the higher order assembly
of collagen-mimetic peptide triple helices at a molecular level, we hope to obtain unique collagen-based
materials on demand for a range of applications.
POST 12-145
Live Cell Imaging of Molecular Conformations and Actions
Peter Yingxiao Wang
Bioengineering, UC San Diego, La Jolla, California, US
Signaling molecules and their activities are well coordinated in space and time to regulate cellular
functions in response to mechanical and chemical microenvironment. Based on fluorescent resonance
energy transfer (FRET), we have developed genetically encoded biosensors to monitor the dynamic
molecular activities (such as Src and FAK activities) in live cells at subcellular compartments when cells
interact with their neighbors or mechanical/chemical microenvironment. In a recent study, we show that a
ubiquitous signaling protein, Src Homology 2 (SH2) Domain-containing Protein Tyrosine Phosphatase 2
(Shp2), displayed unexpected plasticity of conformational changes via intramolecular interactions within
Shp2 (cis-interaction). Utilizing Shp2 biosensors based on fluorescence resonance energy transfer (FRET),
we found that two phosphorylated regulatory tyrosines upon stimulated phosphorylation can compete for
the cis-interaction of the same SH2 domain within Shp2 to achieve plasticity. The antagonistic
combination of contextual amino acid sequence and position (e.g. favorable position combined with
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POSTER ABSTRACTS
adverse sequence) can create a relatively small difference between the two phosphorylated tyrosines in
their overall competitiveness for cis-interaction. Enlarging this difference by swapping the sequences at
the two tyrosine positions resulted in loss of conformational plasticity and reprogrammed downstream
ERK signaling dynamics. Thus, while the combinatorial effect of specific sequence and position of DNA cisregulatory elements on tuning gene expression has been well studied, our results unraveled a new and
simple strategy to achieve cis-regulatory plasticity of protein conformation by coordinating the
combination of sequence and position. We suggest that this strategy can serve as a general and basic
design principle for natural and synthetic proteins, with their conformations and functions tunable by cisinteraction to regulate downstream physiological consequences. These proteins with plasticity can serve
as programmable building blocks or nodes for higher order molecular machines and networks.
POST 12-146
Protein Design: Preventing Protein Aggregation In Recombinant Erythropoietin
Manuel A. Carballo-Amador, Jim Warwicker, Alan J. Dickson
Faculty of Life Sciences, University of Manchester, Manchester, UK
Protein design is a powerful approach for improving protein physicochemical properties with, potential
consequences for stability, activity, and solubility of proteins. Protein solubility characteristics are
important determinants of the success for recombinant therapeutic proteins in relation to expression,
purification, storage and administration. Based on protein structure, an algorithm has been developed to
predict protein solubility, defining polar and non-polar patches on protein surfaces. Using this algorithm,
we predicted amino acid changes that would facilitate expression of forms of human erythropoietin
(rHuEPO) of directed solubility in E. coli. We found that single point mutations (changing a single amino
acid from positive to negative charge) verified the predicted effect on rHuEPO solubility (experimentally
defined as the distribution between soluble and inclusion body fractions) in E. coli. Further application of
this algorithm could provide a valuable tool in the design and engineering of proteins, with enhanced
solubility and stability.
POST 12-147
Intracellular Regulation of the NFE2L3 Transcription Factor
Meenakshi B. Kannan, Volker Blank
Experimental Medicine, McGill University, Montreal, Quebec, Canada
Cap ‘n’ Collar (CNC) basic leucine zipper transcription factors play crucial roles in mammalian gene
expression, stress response and cancer. NFE2L3 (NF-E2 related factor 3), also called Nrf3, was identified as
a member of the CNC family. NFE2L3 forms heterodimers with small MAF proteins and binds to MARE
(Maf recognition element) or ARE (Antioxidant response element) consensus sequences. Our laboratory
has generated mice lacking the Nfe2l3 gene and showed that the knockout animals are highly sensitive to
treatment with the carcinogen benzo[a]pyrene, leading to increased lymphoma formation, suggesting a
tumor suppressor-like function in this context. Contrastingly, in breast cancer cells, we observe increased
invasiveness in the absence of NFE2L3, which illustrates oncogene-like properties of NFE2L3. At the
cellular level, we found that NFE2L3 exists in three different forms (A, B and C). The ‘A’ form is localized in
the endoplasmic reticulum and is N-glycosylated, whereas the ‘B’ form, the full form of NFE2L3, is
predominantly present in the cytoplasm and is also found in the nucleus, upon over-expression. The 'C'
form of NFE2L3 is cleaved at the N-terminus and is localized in the nucleus. I have identified a functional
bipartite nuclear localization sequence (NLS) in NFE2L3 that is required for its nuclear import. It is also
necessary for it to transactivate its downstream targets. In contrast to wt NFE2L3, the NLS mutant fails to
promote invasion in breast cancer cells. Cycloheximide studies revealed a rapid turnover of NFE2L3. Mass
spectrometry and other biochemical analyses revealed ubiquitination and degradation of the NFE2L3
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protein. My results also show the involvement of the ubiquitin ligase and tumour supressor, FBW7, in the
degradation of NFE2L3. Together, my research will elucidate crucial regulatory mechanisms governing the
NFE2L3 transcription factor and determine their significance in NFE2L3-mediated carcinogenesis.
POST 12-148
Phosphorylation and Binding Partners of the NFE2L3 Transcription Factor: Insights into its Role in
Oncogenesis
Isadore Dodard-Friedman, Volker Blank
Experimental Medicine, McGill University, Montreal, Quebec, Canada
Phosphorylation and Binding Partners of the NFE2L3 Transcription Factor: Insights into its Role in
Oncogenesis Post-translational modifications play a major role in the regulation of protein function. Our
laboratory has analyzed the NEF2L3 (NF-E2-like factor 3) protein, which is a member of the Cap 'n' Collar
(CNC) transcription factor family. We found that NFE2L3 is a stringently regulated and post-translationally
modified protein with a high turnover. Moreover, we discovered that NFE2L3 exists in the cell as three
distinct forms located in different cellular compartments which we have called ‘A’, ‘B’ and ‘C’. Our cellular
and mouse studies revealed that its function is linked to carcinogenesis and invasiveness, respectively.
While strong evidence suggests it is glycosylated and ubiquitinated, the phosphorylation status of this
protein is yet to be elucidated. Intriguingly, stringent in silico kinase screens have revealed a series of
potential phosphorylation sites within this protein's transactivation domain. In order to better characterize
NFE2L3 phosphorylation sites, a GST tagged NFE2L3 fusion protein comprising the transactivation domain
was used as a substrate in a kinase screen developed by Screaton and colleagues. Out of the 420 kinases
assessed, five were found to phosphorylate NFE2L3 including the cell cycle kinase PLK1. Phosphorylation
of NFE2L3 by PLK1 was further confirmed by in vitro kinase assays. Furthermore, site directed mutagenesis
of potential PLK1 phosphorylation sites converting respective serines to alanines was performed. In
parallel, immunoprecipitation together with mass spectrometry analysis was used to identify potential
NFE2L3 protein partners. These experiments uncovered a series of potential interactors. Of these, the
golgi protein golgin-84 was confirmed to be a bona fide protein partner of NFE2L3 as confirmed by
coimmunoprecipation and immunoblot experiments. Reverse immunoprecipitation revealed that only the
‘A’ form of NFE2L3 interacts with golgin-84. This data suggests the transcription factor may be processed
in the golgi. Together, my research will lead to a better understanding of the pathways regulating NFE2L3
and linking it to important cellular functions.
POST 12-149
An Orthogonal Genetic System for Rapid Evolution
Chang Liu
Biomedical Engineering and Chemistry, University of California at Irvine, Irvine, California, US
1
We recently developed an orthogonal DNA replication system in the yeast Saccharomyces cerevisiae. This
system consists of an orthogonal DNA plasmid–DNA polymerase pair wherein the orthogonal DNA
polymerase stably and specifically replicates the orthogonal plasmid in the cytoplasm of yeast. Engineered
error-prone DNA polymerases show complete mutational targeting in vivo: per-base mutation rates on
the plasmid can be increased substantially with no increase in genomic rates. Orthogonal replication
therefore serves as a platform for in vivo continuous evolution of user-selected genes and as a system
1
whose replicative properties can be manipulated independently of the host's. A Ravikumar, A Arrieta, CC
Liu. An Orthogonal DNA Replication System in Yeast. Nature Chemical Biology, 10, 175-177 (2014).
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POST 12-150
A Recognition Model Of ACP-HCS Interaction For Programmed Beta-Branching In Type I Polyketide
Synthases
Rohit Farmer1, Anthony S. Haines1, Matthew . Crump2, Christopher . Thomas1, Peter J. Winn1
1
School of Biosciences, University of Birmingham, Birmingham, United Kingdom, 2School of Chemistry,
University of Bristol, Bristol, United Kingdom
Polyketide synthases (PKSs) are enzyme complexes that synthesise a wide range of natural products of
medicinal interest, notably a large number of antibiotics. Type I polyketide synthases can introduce betacarbon branches into a growing polyketide chain via enzymes encoded by the “HMG-CoA synthase (HCS)
cassette”. One of the first polyketide biosynthesis cluster in which the HCS cassette was discovered is
responsible for the synthesis of the antibiotic mupirocin byPseudomonas fluorescens. MupH is the HMGCoA synthase homologue responsible for β-branching in the mupirocin synthesis pathway. To understand
better what allows the HCS cassette to recognise β-branch-associated acyl carrier proteins (ACPs) of the
mupirocin synthesis pathway, we have computationally docked the modelled MupH with the NMR
structure of ACPs. The docking results were also supported by the evolutionary trace data and the
physical properties of the interface residues. Hidden Markov models (HMM) were used to classify ACPs as
branching and non-branching. HMM analysis highlighted essential features for an ACP to behave like a
branching ACP. Through modelling and mutagenesis we identified helix III of the ACP as a probable
anchor point of the ACP–HCS complex. The position of this helix is determined by the core of the ACP and
substituting the interface residues modulates the interaction specificity. Our method for predicting βcarbon branching lays a basis for determining the rules for ACP-HCS specificity and expands the potential
for engineering new polyketides.
POST 12-151
Engineering Photo-Control Of Translation Initiation Using Photoactive Yellow Protein Hybrids
Anil Kumar1, Anna S. Jaikaran1, Alaji Bah2, 3, Julie Forman-Kay2, 3, G. Andrew Woolley1
1
Chemistry, University of Toronto, Toronto, Ontario, Canada, 2Biochemistry, University of Toronto,
Toronto, Ontario, Canada, 3Hospital for Sick Children Research Institute, Toronto, Ontario, Canada
Photo-control of translation initiation could be a powerful tool for probing the role of translational
processes in cellular biology. Translation initiation in eukaryotic cells is regulated by 4EBP (eukaryotic
initiation factor 4E-binding protein). If 4EBP activity can be disrupted by light-induced conformational
changes propagated from a linked photoactive yellow protein (PYP) domain, this may enable photocontrol of cap-dependent translation initiation. Here, we report designs and initial characterization of
4EBP-circularly permuted photoactive yellow protein (cPYP) chimeras. Three constructs were prepared,
54
60
one with only the primary binding site ( YXXXXLΦ : cPYP-4EBP-C0) and two longer constructs also
78
82
containing the secondary binding site ( IPGVT : cPYP-4EBP-C1, cPYP-4EBP-C1N1). All designed chimeras
were expressed in E.coli and are highly soluble. UV-Vis spectra of the dark adapted and irradiated state
confirm the photoswitchable properties of all designed constructs. Recovery rates were increased in the
presence of 4E, consistent with coupling of conformational changes associated with the photocycle and
4E binding. Initial activity using an in vitro translation system based on HeLa cell extracts shows
differential inhibition of translation by the constructs.
POST 12-152
Gold Decorated Peptide Amphiphile Templates for Directed Silver Nanorods Growth
Shlomo Zarzhitsky1, 2, Hanna Rapaport2, 1
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POSTER ABSTRACTS
1
ILSE Katz Center for Meso-and Nanoscale Science and Technology, Ben-Gurion University, Beer-Sheva,
Israel, 2Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion
University, Israel
Peptides are versatile building blocks that can be designed to assume particular structure with respect to
predetermined function and biological activity. Tailoring the exact sequence of amino acids in peptides
allows fine tuning of mechanical and chemical properties, along with self-assembly kinetic. Amphiphilic
and anionic β-sheet peptides may form hydrogels at near physiological pH values. Our group has
previously studied peptides with the amphiphilic motif (Phe-X) in peptides such as Pro-Glu-(Phe-Glu)5Pro. In the current study we were intrigued to explore whether an amphiphilic tripeptide, exhibiting one βpleated motif, i.e. Phe-X-Phe with X = hydrophilic amino acid can be triggered to self-assemble into bsheet fibrils. Relying on the crucial role of aromatic stacking interactions as the driving force in molecular
self-assembly, we explored the self-assembly of this family of peptides with X = Glu\Lys\Cys\Thr. All these
peptides were found to constitute self-supporting hydrogels under various conditions. During the
assembly process FEF peptides were found to spontaneously stack in elongated fibrils composed of βpleated bilayers with preferred angular orientations between intersecting and stemming ones. These
arrangements point to the influence of the phenylalanine aromatic moieties on the intermolecular
interactions of neighboring fibrils. To explore the electrostatic potential activity of Glu residues, fibrils
were exploited as templates for anchoring gold nanoparticles serving as nucleation seeds for directed
mineralization of silver nanorods.
POST 12-153
Engineered Oligosaccharyltransferases With Greatly Relaxed Acceptor Site Specificity
Anne A. Ollis, Sheng Zhang, Matthew P. DeLisa
Cornell University, Ithaca, New York, US
The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (Nlinked) glycosylation and serves as the archetype for bacterial N-glycosylation. This machinery has been
functionally transferred intoEscherichia coli, thereby enabling convenient mechanistic dissection of the Nglycosylation process in this genetically tractable host. For instance, glycosylation-competent E. coli were
used to demonstrate that the primary consensus site for glycan attachment recognized by the bacterial
oligosaccharyltransferase (OST) PglB is (D/E-X-1-N-X+1-S/T; X-1, X+1 ≠ P). In contrast, N-glycosylation site
selection by eukaryotic OST is less specific, requiring only a short N-X-S/T acceptor sequence. Here, we
sought to identify the sequence determinants in PglB that govern its requirement for a negatively charged
residue at the -2 position and effectively restrict bacterial glycosylation to a narrow set of polypeptides.
This involved creation of a reliable genetic reporter for N-glycosylation in E. coli named glycoSNAP
(glycosylation of secreted N-linked acceptor proteins) that was subsequently used to
discover C. jejuni PglB variants with significantly relaxed substrate specificities. Several PglB variants were
identified that no longer obeyed the “minus two rule”, glycosylating an array of noncanonical acceptor
sequences including one in a eukaryotic N-glycoprotein. Taken together, these results underscore the
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POSTER ABSTRACTS
utility of the glycoSNAP assay for shedding light on poorly understood aspects of N-glycosylation and for
engineering designer N-glycosylation biocatalysts.
POST 12-154
The Dynamic Peptide Recognition And Stabilization Mechanism Of Human Leukocyte Antigen
B*35:01
Saeko Yanaka1, Takamasa Ueno3, Kouhei Tsumoto2, Kenji Sugase1
1
Suntory Foundation for Life Sciences Bioorganic Research Institute, Osaka, Japan, 2Department of
Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan, 3Center for AIDS
Research, Kumamoto University, Chuuoo, Japan
In an immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various
antigenic peptides to cytotoxic T lymphocyte (CTL). The stability and long-lived presentation of the
peptide-HLA complex (pHLA), important for efficient antigen-specific CTL activation, highly depends on
bound peptides. Crystal structures of pHLA, however, are very similar to each other irrespective of the
peptides. Thus, the inherent determinant for pHLA stabilization and peptide recognition remains elusive.
In this study, we have examined the mechanism by which HLA-B*35:01 recognizes various peptides and
stabilizes the complex by elucidating its conformational dynamics using relaxation dispersion NMR
spectroscopy. HLA-B*35:01 is one of the most investigated HLAs in the light of the relationship between
antigenic peptide and CTL activity. The NMR experiments of three pHLAs with different peptides showed
that the peptide binding-domain in pHLAs fluctuates between the major and minor conformation in
solution, and that the transition to the minor conformation is accompanied with a negative change of
heat capacity ΔCp, indicating the more dehydrated well-packed conformation of the minor state.
Interestingly, the minor populations detected by the relaxation dispersion experiments correlated well
with the previously reported CTL activity duration. Taken all results together, we revealed that pHLA
loosely recognizes various peptides with a highly identical conformation, and transiently forms a more
dehydrated minor conformation in which the peptide is more tightly bound, resulting in circumvention of
pHLA disintegration. We propose the dynamic recognition-stabilization mechanism as “the transient
induced fit model”.
POST 12-155
Engineering Ordered Protein Assemblies by Helix-Fusion Strategy
Yen-Ting Lai, Todd O. Yeates
Chemistry and Biochemistry, UCLA, Los Angeles, California, US
Many natural proteins exhibit self-assembling properties. Large and ordered architectures, such as viral
capsids, ferritins, clathrin coats, microtubules and bacterial S-layer can arise by repeated interactions
between identical protein building blocks. Designing novel proteins that are capable of self-assembling
into large and ordered architectures similar to those observed in Nature is a challenging task for protein
engineers. Successfully designed protein assemblies with novel properties can lead to important
applications in nanotechnology, material science and medicine. To design novel protein assemblies, the
most critical factor is to fulfill the underlining symmetries of the target assemblies. For example, if one
would like to engineer novel assemblies similar to viral capsids, then the icosahedral symmetry (point
group 532) must be satisfied. Several strategies have been developed in the past decade to engineer
protein building blocks that can self-assemble into highly ordered architectures; the most successful of
these have been centered on the principles of symmetry. One strategy proposed by our group is to fuse
natural protein homo-oligomers with a helix linker in a pre-determined orientation. Through careful in
silicoselection, fusion protein molecules capable of forming structures with higher-order symmetries can
be generated. In my presentation, I will discuss practical considerations that are essential for success in
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the helix-fusion strategy. Successfully engineered tetrahedrally-shaped protein cages (16 nm in diameter)
were verified by x-ray crystallography, small-angle x-ray scattering and electron microscopy. We have also
created an even larger, highly-porous protein framework in the shape of a cube (20 nm in diameter) with
underlying octahedral symmetry, and verified its structure by x-ray crystallography. These structural
studies, however, also highlight that protein cages and frameworks engineered in this way tend to be
flexible and can deviate considerably from perfect symmetry. Modifications to the original strategy, such
as introduction of secondary contacts, can potentially improve the success rate of this method. 1. J. E.
Padilla, C. Colovos, T. O. Yeates, Proc Natl Acad Sci U S A (2001) 98, 2217 2. Y.-T. Lai, D. Cascio, T. O.
Yeates, Science (2012) 336(6085), 1129 3. Y.-T. Lai, K.-L. Tsai, M. Sawaya, F. Asturias and T. O.
Yeates, JACS (2013) 135(20), 7738
POST 12-156
Total Synthesis And Chaperone-Mediated Folding Of A 312-Residue Mirror-Image Enzyme
Michael T. Jacobsen1, Matthew T. Weinstock1, 2, Michael S. Kay1
1
University of Utah, Salt Lake City, Utah, US, 2Synthetic Genomics, La Jolla, California, US
Non-natural D-proteins (composed of D-amino acids) are intriguing macromolecules. First, they are
insensitive to natural proteases, making them appealing drug candidates. Second, they can facilitate
structural analysis via racemic protein crystallography. Third, they possess identical physicochemical
properties to their natural counterparts, making it possible to study otherwise toxic proteins in mirror
image. Unfortunately, D-proteins can only be produced synthetically, hindering production. Furthermore,
folding large and complex D-proteins will be challenging given the absence of D-chaperones. We
hypothesized that a natural chaperone could fold a D-protein, which we addressed by synthesizing a
model client protein in both L- and D-chirality and evaluating its folding by a natural chaperone. We
selected dapA as a model client of the GroEL/ES chaperone. dapA is an essential enzyme in lysine
production and bacterial cell wall synthesis, and its in vivo folding strictly depends on GroEL/ES. The
syntheses of 312-residue dapA in both L- and D- (the longest reported synthetic proteins) were achieved
using solid-phase peptide synthesis with convergent assembly via peptide hydrazides ligations. Rational
mutagenesis and highly optimized synthesis and chromatography methods were necessary to complete
this challenging synthesis. The fully assembled synthetic dapA proteins possess the correct molecular
weight and structure (measured by CD), and they have similar activity to the recombinant enzyme.
Importantly, both L- and D-dapA could be successfully refolded to a similar degree by GroEL/ES. These
results offer a fundamental insight into chaperone mechanism: GroEL/ES recognizes and folds client
proteins through non-specific hydrophobic interactions—activity that even extends to D-proteins.
Because of this ambidextrous folding activity, a new tool (natural GroEL/ES) is now available to assist in Dprotein production. Ultimately, we aim to use the tools and strategies described here to produce a
synthetic D-ribosome for routine production of D-proteins. Highly optimized synthesis strategies are
needed to produce ribosomal D-proteins. This work suggests that their complex folding and assembly
may be facilitated by natural chaperones. The D-ribosome will also serve as a critical springboard to the
creation of mirror-image organisms.
POST 12-157
Creating Self-Assembling Stimulus-Responsive Hydrogels From Protein Components
Danielle Williams1, Ashley Schloss1, Lynne Regan1, 2, 3
1
Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, US, 2Chemistry, Yale
University, New Haven, Connecticut, US, 3Integrated Graduate Program in Physical and Engineering
Biology, Yale University, New Haven, Connecticut, US
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POSTER ABSTRACTS
Stimulus-responsive hydrogels are promising vehicles for the controlled delivery of small molecules, cells,
and other molecular cargo into the body. We have previously demonstrated the formation of selfassembling hydrogels from tetratricopeptide repeat (TPR) protein arrays that form non-covalent crosslinks with corresponding peptides upon mixing. This binding interaction is both pH- and ionic strengthdependent, allowing for encapsulation and subsequent release of cargo in response to a stimulus. The
properties of this interaction directly translate to the macroscopic properties of the gel. Here we present
the design of stimulus-responsive hydrogels made entirely of protein components. The modular nature of
TPR domains allows for the engineering of interactions with different binding affinities/specificities that
encode unique macroscopic properties. Original designs used functionalized star PEGs as the cognate
peptide “cross-linkers” for the TPR arrays. Through the adaptation of SpyTag/SpyCatcher technology, we
have developed a novel concatenation and branching scheme that eliminates the need for functionalized
PEG. We can now create branched protein topologies with multivalent peptides that can be readily
expressed in E. coli with the possibility of all proteins being monodisperse. We will discuss the properties
of various different hydrogels created using this facile and robust production method and how they can
be fine-tuned for biomedical applications.
POST 12-158
Characterization Of A Novel Synthetic Biomaterial For Protein Immobilization
Carrie Marean-Reardon1, Patrick Reardon2, Thomas Squier3, Kathleen McAteer1
1
Washington State University, Richland, Washington, US, 2Pacific Northwest National Laboratory,
Richland, Washington, US, 3Western University of Health Sciences, Lebanon, Oregon, US
Enzymes catalyze a wide variety of processes that are important to environmental, industrial, and
medical applications. However, many enzymes are inherently fragile, requiring specific conditions in
which to operate. Polyethylene glycol diacrylate (PEGDA) is a thiol-reactive material that forms
hydrogels and can be conjugated to cysteine-containing proteins to incorporate a usable binding site
within the gel. In this case, the binding protein incorporated was calmodulin. The characterization of this
method of protein immobilization was undertaken using a small protein, GB1, tagged with the M13
affinity peptide of calmodulin. Solution-state 15N-HSQC NMR spectra were collected on these gels and
show the protein maintains its conformation. The spectra were consistent between free protein in
solution and immobilized protein. The binding is reversible with the addition of EGTA, which chelates
the calcium that the calmodulin needs to maintain binding. These results demonstrate that PEGDA
hydrogels are a viable protein immobilization tool.
POST 12-159
Protein Fragment Exchange: Converting an Arbitrary Binding Protein into a Robust FRET Biosensor
Huimei Zheng1, Jing Bi2, Mira Krendel2, Stewart N. Loh1
1
Biochemistry & Molecular Biology, State University of New York - Upstate Medical University, Syracuse,
New York, US, 2Cell & Developmental Biology, State University of New York - Upstate Medical University,
Syracuse, New York, US
Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial
and temporal distribution of specific molecules in living cells. A major challenge facing biosensor
development is how to functionally couple a biological recognition domain to an output module so that
the binding event can be transduced to a visible and quantifiable signal (e.g. Förster resonance energy
transfer, or FRET). Most designs achieve coupling by means of a binding protein that changes
conformation upon interacting with its target. This approach is limited by the fact that few proteins
possess such natural allosteric mechanisms, and for those that do, the conformational change is
frequently not extensive enough to produce a large distance change between FRET donor and acceptor
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POSTER ABSTRACTS
groups. Here, we introduce protein FRagment EXchange (FREX) to address both problems. FREX employs
two components: a folded binding protein and a fragment duplicated from it, the latter of which can be
chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a
conformational change in which the fragment exchanges positions with the corresponding segment of
the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment
reduces FRET of the unbound sensor to near zero, resulting in a ratiometric FRET response that is
expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin
III, a monobody binding scaffold that can be tailored to recognize multiple targets. Sensors labeled with
Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and
in serum. A genetically-encoded variant of this sensor is shown to be functional in mammalian cell
cultures. This work highlights a novel approach, FREX, which converts an arbitrary binding protein into a
biosensor with high-output, ratiometric FRET response.
POST 12-160
Design of an Albumin-Binding Human Protein by Mimicking the Contact Surface of a Bacterial
Albumin-Binding Domain
Satoshi Oshiro1, Shinya Honda1, 2
1
Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan, 2Biomedical
Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba,
Ibaraki, Japan
Attachment of a bacterial albumin-binding protein module is an attractive strategy to prolong the plasma
residence time of protein therapeutics. However, an antigenicity of bacterial albumin-binding module in
mice was previously reported. Thus, the induction of an unfavorable immune reaction by bacterial
albumin binding module-fused protein or peptide is concerned.
To address this issue, we designed
an alternative albumin-binding protein by imparting albumin-binding affinity to a human protein, using
molecular surface grafting technique. We conducted molecular surface grafting by transferring key
residues of a bacterial albumin-module, Finegoldia magna protein G-related albumin binding domain
(GA) module onto human derived 6 helix-bundle proteins. We utilized structural information (e.g. the
calculation of Accessible surface area of mutation site and the estimation of folding energy change upon
mutation) to avoid the deleterious mutation in molecular surface grafting.
As a result, one of the
designed proteins specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM).
Despite 13–15 mutations, the designed proteins maintain the original secondary structure by virtue of
careful grafting based on structural information. Competitive binding assay and thermodynamics analysis
showed that the best binder have similar HSA-binding site of GA module.
These results indicate that
the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular
surface grafting in combination with structural informatics is an effective approach for avoiding
deleterious mutations on a target protein and for imparting the binding function of one protein onto
another. Reference Oshiro, S. and Honda, S. Imparting Albumin-Binding Affinity to a Human Protein by
Mimicking the Contact Surface of a Bacterial Binding Protein” ACS Chemical Biology, 9 (4), 1052–1060
(2014)
POST 12-161
Developing Soluble Co-Receptor Mimetics For The Study Of HIV Env/Receptor Interactions
Agnes Hajduczki, Virgilio Bundoc, Edward A. Berger
National Institutes of Health, Bethesda, Maryland, US
129
POSTER ABSTRACTS
The HIV envelope glycoprotein (Env) mediates virus entry by initiating fusion of the viral envelope with the
cell membrane upon receptor engagement. The surface-exposed gp120 subunit is a dynamic molecule
that undergoes a series of stepwise conformational changes in response to interactions with the primary
receptor CD4, and co-receptors, CCR5 or CXCR4. Obtaining structural information on the various
intermediates during viral entry is a key focus of antiviral and vaccine research and could open the doors
for more effective treatment and prevention. The co-receptors are G-protein-coupled receptors, anchored
with seven membrane-spanning helices, leaving the N-terminus and three extracellular loop regions
exposed outside the plasma membrane and available for interactions. Due to the inherent insolubility of
membrane proteins, working with the intact co-receptors outside the context of the membrane is not a
viable option. This project aims to develop recombinant soluble co-receptor mimetics featuring critical
determinants of CCR5 using two approaches: fusing the CCR5 moieties to soluble CD4 by flexible
polypeptide linkers and using a globular, stable scaffold protein to orient the co-receptor determinants in
an optimal conformation to recreate the binding interaction with gp120. We have successfully
overexpressed and purified the recombinant proteins from mammalian cells. Characterization of the
gp120-binding properties of the variants is underway using a vaccinia-based cell fusion assay where
soluble CD4 has been shown to induce membrane fusion between Env-expressing effector cells and
target cells bearing CCR5, but no CD4. When the soluble CD4 fused co-receptor mimetic is added to the
fusion reaction, there is strongly impaired activation of membrane fusion compared with sCD4 alone,
suggesting that the CCR5-derived portion of the protein competes with cell-surface CCR5 for binding to
gp120. Conversely, the mimetic displays stronger neutralization activity than sCD4 in a standard fusion
assay. The soluble variants of CCR5 will be used to elucidate the conformational changes in gp120 that
immediately precede membrane fusion, and potentially for collaborative high-resolution structural
analyses of the gp120-coreceptor complex.
POST 12-162
Constructing Highly Detectable Fluorescence Reporter Protein Forin Vitrosingle Molecular
Screening
Kotaro Nishiyama1, Norikazu Ichihashi1, 2, Yasuaki Kazuta1, 2, Tetsuya Yomo1, 2
1
Osaka Univeristy, Suita, Japan, 2Exploratory Reasearch for Advanced Technology Japan Science and
Technology Agency, Suita, Osaka, Japan
Recently, as the improvement of micro-compartment technology, biochemical reactions are reconstructed
inside micro-compartments such as lipid vesicles. To detect the gene expression inside microcompartments, reporter proteins that produce fluorescence are widely used. The fluorescence proteins
such as GFP have problem in the strength of signal (Liu HS et al., 1999, Lang et al., 2006) comparing to
enzymatic reporter proteins such as β-galactosidase(β-Gal) (Yu H et al., 2003), which keep degrading
substrate and increasing signal. β-Gal is widely used because of its strong substrate degradation reactivity
and variety of substrates (Zabin Z et al., 1975). β-Gal becomes active by forming tetramer. When it loses
its amino terminus (e.g. residue position 23-31, called ω-protein), cannot form tetramer, and become
inactive dimer. This dimer recovers its reactivity by binding with protein which complements lost residue
(called α-protein). This complementation is called “a-complementation” and famous as blue-white
screening. In in vitro, it has been used to detect RNA self-replication reaction in liposomes (Nishiyama et
al., 2012) because short size of a-protein is advantageous for RNA replication reaction. However,
enzymatic activity of a-complementation is weaker than that of b-galactosidase in in vitro. In this study,
we attempted to improve the α-protein activity by optimizing the α-protein size and also by the liposomebased directed evolution (Nishikawa et al., 2012).
[Result] We perfomed liposome-based directed
evolution method for α-protein. In this method, we first performed random mutagenesis of the DNA
encoding α-protein, and then encapsulate the DNA in liposome with cell-free translation system under the
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POSTER ABSTRACTS
condition of 1 DNA molecule per liposome. After incubation at 37°C, collected the liposomes which
volume is 10 fL and show stronger fluorescence intensity than certain threshold by using a fluorescence
activated cell sorter (FACS). We then amplified the collected DNA by PCR and encapsulated the DNA into
liposome again. We repeated this cycle for 23 times and got mutant which show 1.7 times higher acomplementation activivty than that of wild type. To further improve the activity, we then searched the
optimum size of α-protein . We constructed α-protein of various size ranging 81 bp to 687 bp, and found
that 180 bp (alpha-180), shoter than the original one (570bp), exhibit the highest acitivy.
POST 12-163
Model Building Of Antibody-Antigen Complex Structures Using GB/SA Scores
Narutoshi Kamiya1, Noriko Shimba2, Haruki Nakamura1
1
Osaka University, Suita, Japan, 2Panasonic Corporation, Seika-cho, Soraku-gun, Japan
A structure prediction method for antibody (Ab) and antigen (Ag) complexes, which consists of conformer
generation of Ab and Ag, molecular docking to make a decoy set, and a ranking the set using a scoring
function, is one of the most important technics to support the design of antibody drugs and biosensors.
Since the scoring functions used in the conventional docking programs are not always suitable for Ab and
Ag, the prediction result is not accurate. In addition, there is no evaluation method whether the predicted
complex is real or not. In this work, we present a scoring function and an evaluation method using
molecular dynamics (MD) simulation to improve the accuracy. We introduced a score using the
Generalized-Born/Surface Area method, GB/SA score, where interaction energy between Ab and Ag
including solvation free-energy is calculated. To test the GB/SA score, we executed the ranking of 82 AbAg decoy sets generated by the docking program, SurFit [1]. The area under the curve (AUC) obtained
from the GB/SA score (= 0.985) was better than that using the SurFit score (= 0.882), suggesting that our
score improved the docking accuracy. We also carried out the ranking of decoy sets in the ZDOCK
benchmark, and obtained a better AUC than that using ZDOCK. We used our new MD program, psygeneG [2], which utilizes GPU for acceleration of the electrostatic treatment by our original zero-dipole
summation method [3]. We have attained similar dynamics properties to the particle mesh Ewald method
in membrane protein and DNA-water-ion systems [4, 5]. We executed short 10-ns MD simulations to
evaluate the decoy structures, where the top 8 to 10 decoys from each of three decoy sets were chosen
for trials. During MD, all native-like decoys kept each initial complex structure, and some (not all) nonnative decoys dissociated. We calculated an average GB/SA score over the last 6-ns MD trajectories. All of
near-native decoys exhibited favorable scores, and most of non-native decoys did not. Thus, our approach
combining SurFit and short MD with GB/SA scores is effective to build correct Ab-Ag
models. References [1] Kanamori E. et al. 2013. In Biomolecular forums and functions pp. 160. World
Scientific, Singapore. [2] Mashimo T. et al. J. Chem. Theory Comput. 9, 5599 (2013). [3] Fukuda I. et al. J.
Chem. Phys. 134, 164107 (2011). [4] Kamiya N. et al. Chem. Phys. Lett. 568, 26 (2013). [5] Arakawa T. et
al. PLoS ONE 8, e76606 (2013).
POST 12-164
Engineering Novel Phosphopeptide Recognition Modules that Recognize Targets In Vitro and in E.
coli
Nicholas Sawyer1, 2, Lynne Regan1, 2, Brandon Gassaway3, 4, Jesse Rinehart3, 4, Adrian Haimovich3, 5, Farren
Isaacs3, 5
1
Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven,
Connecticut, US, 2Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut,
US, 3Systems Biology Institute, Yale University, West Haven, Connecticut, US, 4Cellular and Molecular
131
POSTER ABSTRACTS
Physiology, Yale University, New Haven, Connecticut, US, 5Molecular, Cellular and Developmental
Biology, Yale University, New Haven, Connecticut, US
Protein phosphorylation is a widespread biological mechanism for cellular adaptation to environmental
changes. Dysregulation of phosphorylation signals is implicated in a wide variety of diseases. Thus,
detection and quantification of protein phosphorylation is highly desirable for diagnostic as well as
academic applications. Here we present a general strategy for detecting phosphopeptide-protein
interactions in E. coli. We first re-design a model tetratricopeptide repeat (TPR) protein and characterize
its interaction with a target phosphopeptide in vitro. We then use this phosphopeptide-protein interaction
to benchmark a split mCherry assembly assay in Escherichia coli. We demonstrate that split mCherry
assembly coupled to fluorescence-activated cell sorting (FACS) can be used to separate highly fluorescent
clones from a mixture of split mCherry peptide-protein pairs. This high-throughput strategy can thus be
used to isolate proteins that specifically recognize a variety of different phosphopeptides.
POST 12-165
N-linked Glycosylation of HIV-1 core gp120 is Not Required for Native Trimer Formation or Viral
Infectivity
Ujjwal Rathore1, Piyali Saha1, Sannula Kesavardhana1, Aditya A. Kumar1, John R. Mascola2, Raghavan
Varadarajan1, 3
1
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India, 2Vaccine Research
Center, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, Maryland,
US, 3Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
Human immunodeficiency virus 1 (HIV-1) is the causative agent for Acquired Immunodeficiency
Syndrome (AIDS). The envelope (env) glycoprotein of HIV-1 is a trimer of the receptor binding soluble
subunit gp120 and the transmembrane gp41 heterodimers. gp120 is the primary viral component which is
exposed on the virion surface and is the main target for neutralizing antibodies. Glycans constitute
approximately 50% of the molecular mass of gp120 and shield env from immune recognition. The
structure of gp120 can be subdivided into three parts, the inner domain, the outer domain (OD) and the
bridging sheet. OD is heavily glycosylated and contains important neutralization epitopes. Glycans are
believed to be indispensable for proper folding of gp120 and viral infectivity. However, the role of
glycosylation in the folding of gp120 has not been rigorously tested. Using rationally designed mutations
and yeast surface display, we show that glycosylation is not essential for in vivofolding of OD alone or OD
in the context of core gp120. Two mutants of core gp120 were isolated, which retained a single inner
domain glycan, and broadly neutralizing antibody (bNAb) binding. In previous studies, whenever multiple
glycosylation sites were mutated in combination, it resulted in loss of viral infectivity which led to the
hypothesis that a certain level of glycan coverage is essential for maintaining infectivity of the virion. In
contrast, the present study shows that virions retain infectivity even in the absence of all glycans from
core gp120. bNAbs against HIV-1 show a very high level of affinity maturation. Germline (GL) reverted
variants of these bNAbs fail to recognize mature env. Thus immunogens based on mature env will likely
not be able to activate the target GL-B cells. Immunogens that bind well to precursors of bNAbs as well as
the corresponding mature antibodies may help to elicit such bNAbs upon vaccination. In the current
study, we show that recognition of a GL-bNAb increases substantially with the progressive loss of glycans
from JRFL E168K pseudoviruses. Glycan free OD domain immunogens bind to mature as well as a GL
reverted variant of bNAb VRC01 with nM affinity. The present results inform immunogen design, targeting
mature as well as GL bNAbs, clarify the role of glycosylation in gp120 folding and illustrate general
methodology for design of glycan free, folded protein derivatives.
POST 12-166
132
POSTER ABSTRACTS
Silicon Transporters: From Membrane Proteins To Nanotechnology
Laura Senior, Sarah Ratcliffe, Michael Knight, Adam Perriman, Stephen Mann, Paul Curnow
Diatoms are abundant and diverse single-celled algae that are sheathed in a silica-coated outer cell wall.
This structure, known as the frustule, forms hierarchical silica structures which are patterned with
nanoscale precision at ambient pH and temperature. In order to form these frustules diatoms must source
silicon from their environment. It is thought that this is done through membrane proteins that specifically
interact with silicon and are known as silicon transporters (SITs). When the SITs are energised by an
electrochemical sodium gradient they transport silicic acid (soluble silicon) into the diatom cell, where it
undergoes polymerisation to form silica. This work has focused on the reconstitution of a recombinant SIT
from the diatom Thalassiosira pseudonana into synthetic liposomes in order to replicate this
biomineralization process. The SIT is recombinantly expressed and purified before being reconstituted
into liposomes that contain biomineralizing peptides. The transporter is then energised by applying a
transmembrane sodium gradient so that it actively pumps silicic acid from the outside to the inside of the
proteoliposome. The small internal volume of the proteoliposome should cause the transported silicic acid
to rapidly approach saturation; under these conditions the biomineralizing peptides should catalyze and
template the formation of nanostructured silica. This novel method incorporates many of the principles of
diatom biomineralization such as encapsulation and active transport, that are absent in bulk silicification
experiments. Further work will attempt to synthesise novel silica structures by adjusting reaction
conditions or functionalising the silica products by trapping fluorophores, enzymes and other reactive
species within the silica matrix.
POST 12-167
Characterization of Thermotoga maritima Maltotriose Binding Protein
Laura Masson, Jonathan Dattelbaum
Thermotoga martima is a thermophilic, Gram-negative bacteria found primarily in hot springs and
hydrothermal vents. Its thermophilic characteristics mean that proteins found in this bacterium should be
extremely stable, even at very high temperatures, making them ideal for manipulation and biosensor
design. The protein of interest for our project is the T. maritima maltose binding protein (TmMBP), which
is a member of the periplasmic binding protein superfamily. Using site-directed mutagenesis of residues
in and around the binding pocket, we constructed eight single cysteine mutants of TmMBP. The
individual Cys residues will be covalently modified with environmentally-sensitive fluorophores to
interrogate maltose binding to the protein. The altered fluorescence characteristics will be used to
determine a dissociation constant for maltose. We will then use circular dichroism to evaluate the stability
of each mutant. Ultimately, we hope to design a TmMBP biosensor that is stable, can bind molecules
other than maltose, and will fluoresce when the target molecule is bound.
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POSTER ABSTRACTS
POST 12-168
Zinc Induces Self-Assembly Of Bacterial Thermoalkalophilic Lipases: A Strategy For Thermostability
Emel Timucin, Osman U. Sezerman
Sabanci University, Istanbul, Turkey
Bacterial thermoalkalophilic lipases are both thermostable and aggregation-prone. Moreover, these
lipases possess a unique zinc site. Zinc ions have been shown to be a prevalent cause of protein
aggregation; especially in human diseases affecting the central nervous system. Yet any mechanism
linking zinc and aggregation in the context of thermostability has not been characterized. In this study we
investigated the effects of zinc ion on the aggregation mechanism of the thermoalkalophilic lipase
from Bacillus thermocatenulatus (BTL2). Dynamic light scattering analysis showed that the recombinant
BTL2 was dispersed in dimeric form with a hydrodynamic radius (RH) of 7 nm and when treated with
2+
Zn chelator, it adopted monomeric form (RH=3.6 nm). Although the zinc free BTL2 restored 90% activity
at 25°C, it showed only 50% activity at 50°C, suggesting that zinc is essential for self-assembly which
confers thermostability to BTL2. After 12 hours incubation of BTL2 with 50 µM ZnCl2, it formed ordered
aggregates (RH>27 nm) able to bind congo red. These aggregates showed significant loss in activity (80%)
at 25°C, while they displayed relatively marginal decrease in activity (30%) at 50°C. Although the zinc
induced aggregates showed very low activity at 25°C, most likely due to hindrance of the catalytic site by
aggregation, 50% more activity could be restored from these aggregates at 50°C. This particular
observation implies that increased kinetic energy dissociates aggregates into functional forms. Using CD
spectroscopy we further investigated the impact of temperature on the kinetics of zinc induced
aggregation and found that the rate of aggregation was much slower at 50°C compared with the rate at
25°C (Fig. 1), confirming that increased kinetic energy curtails the zinc induced intermolecular interactions
and impedes with the self-assembly of BTL2. To this end, we suggest that thermoalkalophilic lipases are
prone to self-assemble through interactions mediated by zinc, which can be largely reversed by
temperature switches. Essentially the reversibility of the zinc induced aggregation by high temperatures
has a biological relevance, in which aggregation becomes a concrete preference of thermoalkalophilic
lipases for thermostability. In line with the above findings, we will also discuss the roles of hydrophobic
amino acids on the mechanism of zinc induced aggregation through a mutation (W211A) located in close
proximity (~5 Å) to zinc.
POST 12-169
The SasG E-G5 Protein Fold Forms A Stable Contiguous Rigid Nanorod Of Tunable Length
Fiona Whelan1, Dominika T. Gruszka2, Jane Clarke2, Jennifer R. Potts1
1
Department of Biology, The University of York, Heslington, North Yorkshire, United
Kingdom, 2Department of Chemistry, Cambridge University, Cambridge, United Kingdom
Every year, infections associated with implanted medical devices affect millions of patients worldwide,
resulting in significant morbidity, mortality and high costs to health services. Central to the antibiotic
resistance and persistence of these infections is the formation and repeated dissemination of bacterial
communities termed biofilms. Protein-dependent biofilm formation is mediated by SasG in S.
aureus. SasG comprises an N-terminal functional domain implicated in bacterial adherence that is
projected from the cell surface by a series of near identical sequence repeats, terminating in a covalent
cell-wall tether. Negative stain EM studies have shown that SasG forms a uniform fringe of fibres
projecting up to 54 nm from the cell surface [1]. We have previously determined the structure of the
minimal repeat sequence, defining a novel G5 fold with an N-terminal extension domain 'E' that form a
highly anisotropic rod-like fold [2]. Models of a repetitive E-G5 fold suggest the protein should form a
stable extended nanorod. We have produced a truncation series of repetitive sequences on biologically
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POSTER ABSTRACTS
relevant scales and performed a range of biophysical and structural analyses including AUC, SEC-MALLS,
QELS and SAXS. These analyses indicate that stepwise addition of E-G5 repeats generates stable, soluble,
contiguous rigid nanorods up to 63 nm in length and only 2 nm in width of tunable length in repeat
increments. 1. Corrigan, R.M., et al., The role of Staphylococcus aureus surface protein SasG in adherence
and biofilm formation. Microbiology, 2007. 153 (Pt 8): p. 2435-46. 2. Gruszka, D.T., et al., Staphylococcal
biofilm-forming protein has a contiguous rod-like structure. Proc Natl Acad Sci U S A, 2012. 109(17): p.
E1011-8.
POST 12-170
Computational Design Of Allosteric Antibody
Olga Khersonsky, Sarel Fleishman
Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel
Allosteric regulation is central to the control of many metabolic and signal-transduction pathways, and
thus it was named “the second secret of life”. The mechanisms of allostery, however, are largely remaining
enigmatic. De novo design of allosteric proteins is particularly challenging due to the need to model the
backbone degrees of freedom, and to define the multiple protein states. Recently, AbDesign algorithm
was developed in our lab for de novo design of antibodies. It is guided by natural conformations and
sequences, and exploits the modular nature of antibodies to generate an immense space of
conformations, which can be used as scaffolds for design of stable high-affinity binders. We have used
2+
AbDesign to design an allosteric antibody, in which Zn is an allosteric effector of fluorescein binding. We
2+
introduced Zn binding site into the H3 CDR of high-affinity fluorescein-binding antibody, with the
2+
assumption that without Zn chelation, the floppiness of H3 backbone would prevent fluorescein from
binding. We used as a template the fluorescein-binding antibody (pdb ID 1x9q), and spliced into it various
2+
H3 segments to obtain a variety of scaffodls. Zn binding site was introduced into these scaffolds with
RosettaMatch, and the sequence was subsequently optimized by design. ~100 designs were
2+
experimentally tested by yeast display for fluorescein binding in the presence or absence of Zn . We have
found that fluorescein binding by 1x9q is very sensitive to any changes and requires atomic accuracy. One
2+
of the designs demonstrated significantly higher fluorescein binding in the presence of Zn , and this
allosteric behaviour is currently being further investigated.
POST 12-171
Development And Optimization Of An e.Coli-based Display Platform For Selection Of Affinity
Proteins
Filippa Fleetwood, Ken Andersson, Stefan Ståhl, John Löfblom
School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden
Combinatorial protein engineering using surface display is commonly used for the generation of specific
affinity proteins. An advantage of cell surface display over other systems such as phage display is the
possibility to use flow-cytometric cell sorting. Autotransporters is a class of surface proteins in Gramnegative bacteria that enable efficient translocation of passenger proteins to the outer membrane. The
aim of this project is to investigate and further develop an autotransporter-based display system
in Escherichia coli for combinatorial protein engineering applications. Potential advantages of this system
include a high surface expression level of the autotransporter AIDA-I, which is beneficial for flowcytometric purposes, as well as a high transformation frequency due to the use of Gram-negative bacteria,
which will allow for large library sizes. Optimization data including evaluation of different promoters, and
selection from a mock library of Affibody molecules will be presented.
135
POSTER ABSTRACTS
POST 12-172
Computational Redesign of Metagenomic Enzymes
Justin B. Siegel
Genome Center, University of California at Davis, Davis, California, US
The development of new computational methods to enable the efficient exploration of protein sequencestructure-function space is critical for the design of enzymes with novel functions. We have developed a
new method to explore the structurally relevant sequence-space of an enzyme family, and identify protein
sequences that can be used as novel starting points for enzyme engineering. This method takes
advantage of the recent explosion of available protein sequences derived from genomic data. Within an
enzyme family there are now commonly thousands of available protein sequences that are diverse in
sequence and broadly cover the structurally relevant sequence-space for the enzyme family. By modeling
the three-dimensional structure of each “metagenomic enzyme” we can evaluate the active site, and
predict if it can be readily engineered for the specific reaction of interest. We have applied this method to
engineer alpha-keto acid decarboxylases, which resulted in a novel enzyme experimentally validated to be
four orders of magnitude more specific for long chain keto-acids than any enzyme reported to date.
POST 12-173
Accurate Prediction of Protein Stability by Explicit Negative Multistate Design
James A. Davey, Christian K. Euler, Roberto A. Chica
Chemistry, University of Ottawa, Ottawa, Ontario, Canada
Current computational protein design (CPD) methodologies allow for the in silico screening of amino acid
sequences on a scale that is experimentally impossible to achieve. While CPD can reliably enrich
predictions of stable protein sequences, the methodology is currently unable to accurately correlate
predicted and experimentally-validated stabilities of protein sequences. Traditionally, CPD is performed
using a positive design approach whereby sequences are scored and ranked on a model of the folded
protein structure, approximated by either a single fixed protein backbone or a backbone ensemble. In an
attempt to reconcile calculation and experiment, we present the first negative design approach that
utilizes a backbone ensemble as an explicit negative state model. Our negative state model, intended to
approximate the unfolded protein, was created from an off-target backbone ensemble shown to poorly
predict and enrich stable protein sequences. Conversely, our model for the folded protein was created
from an on-target ensemble shown to correctly predict and enrich stable protein sequences. A correlative
2
(R = 0.82) quantitative structure-activity relationship (QSAR) was generated using a training set of 18
known stable Streptococcal protein G domain β1 (Gβ1) sequences. Our QSAR was then applied to the
prediction of stability for 10 new Gβ1 sequences. Experimental validation of the stabilities of these 10 new
2
sequences shows excellent agreement between prediction and experiment (R = 0.77) providing a proofof-concept for the application of off-target backbone ensembles in explicit negative multistate design for
the prediction of protein stability.
POST 12-174
Plastic Protein Design: A Novel Design Algorithm Using Backbone And Side-Chain Ensembles To
Model Protein Flexibility
Christian D. Schenkelberg1, Derek J. Pitman3, Yao-ming Huang2, Christopher Bystroff1
1Department of Biology, Rensselaer Polytechnic Institute, Troy, New York, US, 2Department of
Bioengineering and Therapeutics, University of California, San Francisco, San Francisco, California,
US, 3Department of Biological Chemistry, University of California, Los Angeles, US
Developing and implementing an effective and realistic three dimensional protein model for
computational protein design remains a major challenge in structural biology. It has long been
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POSTER ABSTRACTS
understood that protein design based on a static backbone conformation is not adequate to predict
probable amino acid sequences (Kuhlman and Baker, 2000). Previous attempts at modeling backbone
flexibility have relied on performing design on multiple static conformations of the backbone (Allen and
Mayo, 2010) or introducing backbone movements as part of the protein design move-set, such as in the
backrub algorithm (Smith and Kortemme, 2008). In order to efficiently model protein backbone flexibility
during computational design, we have implemented a design scheme termed “plastic” design which
incorporates elements of these existing backbone flexibility models. Plastic design involves designing a
protein primary sequence on an ensemble of related structures of a single protein where each member of
the ensemble is designed in parallel with the other members, computing a Boltzmann-like weighted
average energy as the score of the design. Ensembles are generated primarily from the backrub modeling
application (Friedland et al., 2009) but can be generated via other existing methods, such as molecular
dynamics. Rotamers are selected in the context of an ensemble generated by rotating all combinations of
the sidechain χ angles by ±10° from a “parent” rotamer derived from the Dunbrack rotamer library
(Shapovalov and Dunbrack, 2011). An appropriate “child” rotamer for each backbone is selected from this
ensemble by finding the lowest energy rotamer from among all the child rotamers in the rotamer
ensemble. We have found that plastic protein design yields predictions that more realistically simulate
protein behavior as modeled by molecular dynamics trajectories.
POST 12-175
Repeat-Protein Directed Synthesis Of Gold Nanoparticles With Tunable Morphology And Optical
Properties
Tijana Grove, Xi Geng
Chemistry, Virginia Tech, Blacksburg, Virginia, US
Fundamental advances in biotechnology, medicine, electronics, and energy require methods for control
of structure and function on nanometer scales. Extensive research has focused on the bio-enabled
synthesis of Au nanoparticles (AuNP) as fundamental building blocks for the construction of catalytic,
optoelectronic, and biosensing devices. However, the mechanism responsible for biomolecule-directed
nanoparticle and nanostructure formation remains unclear. Here we will discuss morphology and optical
properties of AuNPs synthesized in the presence of modular repeat-protein arrays. Rod shaped,
modular consensus sequence tetratricopeptide repeat proteins, CTPRs, containing 3, 6, or 18 tandem
repeats were used in-situ for synthesis of Au nanoparticles in the presence of zwitterionic buffers MOPS,
HEPES, and PIPES. Transmission electron microscopy and optical spectroscopy data indicate that the
protein concentration, shape, and choice of buffer all have implications on Au nanoparticle morphology,
ranging from flowers to spheres. Circular dichroism and fluorescent spectroscopy provide further
information on the molecular interaction between CTPR proteins and both Au precursor and AuNP.
POST 12-176
Designed Affinity Reagents Directed to Heat Shock Protein C-terminal Motifs
Robert Wells, Akiko Koide, Shohei Koide
University of Chicago, Chicago, Illinois, US
Affinity reagents that bind with high affinity and specificity to target proteins are indispensable for
biological research. There is an increasing need for affinity reagents that bind to pre-defined linear
protein motifs, especially those targeted by modular peptide-binding domains, such as SH2, PDZ and
TPR. However, it is generally difficult to recognize such short, flexible peptides with high specificity. To
address this challenge, our group has established a new class of designed binding proteins called ‘affinity
clamps’. An affinity clamp is a two-domain fusion protein, consisting of a ‘primary domain’, chosen from
nature that binds to the desired target, but may have low affinity and/or low sequence specificity. This
137
POSTER ABSTRACTS
primary domain is fused via a flexible linker to an ‘enhancer domain’, which is engineered to bind to the
target-primary domain complex. Together, these domains ‘clamp’ to the target peptide to increase the
interaction surface area, and are able to achieve a dramatic increase in affinity and specificity. Affinity
clamps have been successfully created using PDZ and SH2 primary domains with the FN3 "monobody" as
the evolved enhancer domain. Here, we aim to expand the sequence space of affinity clamp recognition
to charged C-termini. C-termini are ideal targets for selective protein capture, because they are unique
chemical moieties present in every protein, can be easily predicted from genomic data and can be
mimicked by short peptides. As proof-of-principle, we chose the C-terminal sequences of the human heat
shock proteins as affinity clamp targets. They are important for co-chaperone binding and protein
function. Therefore, we envision that these affinity clamps will be unique inhibitors of heat shock protein
function, as well as useful affinity reagents. Using a TPR primary domain, we have generated affinity
clamps with greatly enhanced affinity to the human HSP90 C-terminus, while retaining high
specificity. Further in vitro and in cell characterization are underway. Our results strongly suggest the
feasibility of developing affinity clamps using TPR domains and the generality of the affinity clamp
concept. Supported by the NIH grant R01-GM090324.
POST 12-177
Consensus Design Of A NOD Receptor Leucine Rich Repeat Domain With Binding Affinity For A
Muramyl Dipeptide (MDP), A Bacterial Cell Wall Fragment
Rachael Parker, Ana Mercedes-Camacho, Tijana Z. Grove
Chemistry, Virginia Tech, Blacksburg, Virginia, US
Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their
modular architecture and biophysical properties. Here we present the design of a scaffold based on the
consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate
immune system receptors. Consensus sequence design has emerged as a protein design tool to create de
novo proteins that capture sequence-structure relationships and interactions present in nature. The
multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in
NOD proteins, resulted in a consensus sequence protein containing two internal and N- and C- capping
repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any
affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To
our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant
ligand. CLRR2 will be used as a scaffold to incorporate binding affinity for other nonproteinaceous
ligands.
138
POSTER ABSTRACTS
POST 12-178
Solvent Engineering To Improve Enzymatic Synthesis Of A Sugar-Based Surfactant
Rodrigo A. Arreola-Barroso, Gloria Saab-Rincón
Ingenieria Celular y Biocatalisis, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico
Surfactants are important molecules to maintain pharmaceutic, cosmetic and food products in one phase.
Some alpha-amylases can produce the non-toxic surfactant alkyl-glucoside from the abundant substrate
starch though an alcoholysis reaction, in a single step, when in the presence of alcohols.Thermotoga
maritima enzyme AmyA has been the best alpha-amylse to carry out alcoholysis reactions. This ability has
been improved through amino acid substitution on its sequence. However, further improvement in this
synthetic reaction through media engineering has not been explored. In the present work we explore
reaction media engineering to further improve the yield of the synthetic reaction. Here we show that a
decrease in water activity by a cosolvent is enough to increase the production of alkyl glycosides. We
found that the addition of 2-methyl-2-butanol increases three-fold butyl glycosides synthesis compared
to the condition where only a water-butanol mixture is used. Additionally, a proper cosolvent allows the
alcoholysis reaction to run in one phase when using long chain alcohols, which potentiates the use of
higher alcohol concentrations in the reaction. Our work shows that solvent engineering can be used to
increase the production of alkyl glycosides using alpha-amylases, opening new opportunities for the
improvement of this reaction.
POST 12-179
Delivery Of Natural And Non-Natural Chemical Entities Into Cells Using Anthrax Toxin
Amy Rabideau, Xiaoli Liao, Bradley Pentelute
Chemistry, MIT, Cambridge, Massachusetts, US
Delivery of polypeptides, especially those containing non-natural functionalities, into the cytosol of cells is
a major challenge in the biotechnology field. We explored the capability of protective antigen (PA), the
pore-forming protein of anthrax toxin in the delivery of these entities. To do this, we conjugated various
cargos covalently to the C-terminus of a 263-residue domain (LFN) recognized by PA. We found that PA
can efficiently transport a wide range of natural and non-natural chemical entities including single domain
antibody mimics, small molecules, and peptides with backbone, side chain, and stereochemical
modifications. This platform also enabled the delivery of a functional peptide and protein cargo into the
cytosol of cells that can be used to disrupt specific protein-protein interactions in cancer cells.
POST 12-180
Thermostabilization of Aspergillus oryzae Cutinase
Danielle A. Basore1, 4, Abhijit Shirke2, 4, Glen Butterfoss3, Richard Gross2, 4, Christopher Bystroff1, 5, 4
1
Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 2Chemistry and Chemical
Biology, Rensselaer Polytechnic Institute, Troy, New York, US, 3Center for Genomics and Systems
Biology, New York University, Abu Dhabi, United Arab Emirates, 4Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, US, 5Computer Science,
Rensselaer Polytechnic Institute, Troy, New York, US
Cutinases are esterases produced by various phytopathogenic fungi to break down cutin, the biopolyester
coating found on leaves and fruit. They also are powerful hydrolases that can cleave ester bonds of rigid
polyesters such as poly(ethyleneterephthalate), PET, opening up new options for polymer recycling and
surface modification reactions. To increase the rate at which cutinases can hydrolyze PET, it is useful for
o
the enzyme to function at temperatures above the glass transition which, for PET, is 75 C. To function at
139
POSTER ABSTRACTS
such temperatures there is a need to increase the thermal stability of cutinases. Consequently, work is in
progress to improve the thermal stability of the cutinase from Aspergillus oryzae (AoC) using Rosetta
and other rational design approaches focusing on both surface interactions and core packing to reduce
o
the void space. Wild-type AoC has a Tm of 61 C, an additional disulfide bond not present in other
cutinases and an extended groove near the active site. Of particular interest were two sets variants with
changes in amino acids at the surface of AoC: i) A102D, Q105R, G106E and ii) N133A, S140P, E161T,
o
A166P. These variants increased the Tm by 6 and 4 C, respectively. By combining these 7 mutations the
o
Tm was further increased by 2 C. However, the relative activity was approximately half that of the wild
type AoC. The reasons for this increase in stability and loss in activity are currently under study.
POST 12-181
Beta-Hairpin Tags For Increased Expression Of Helical Peptides
Melissa E. Lokensgard, John J. Love
Many small helical domains can only be expressed at high levels in E. coli when fused to larger amino-
terminal tags (such as Ubiquitin, SUMO, GST, MBP, thioredoxin, etc.) or when cloned in periplasmic
expression vectors. However, StreptococcalProtein G (Gβ1), a 56aa domain, can be easily expressed in
high yield using simple pET cytosolic overexpression systems with IPTG-inducible promoters. Gβ1 has also
been successfully used as an amino-terminal fusion partner for poorly-expressed proteins. Gβ1 and other
tags can improve not only yield, but also solubility and folding of the target protein, and some can double
as handles for chromatographic purification and crystallization. These fusion tags are thought to act by
facilitating or accelerating folding of the fused protein and either stabilizing the folded state or
destabilizing the unfolded state. We have discovered a twelve amino acid fusion tag derived from a betahairpin in Gβ1 that dramatically increases the overexpression yields of three evolutionarily unrelated
helical proteins when fused to either terminus. Deconvoluted circular dichroism spectra show that the
fusion proteins contain beta-character proportional to the added hairpin, suggesting the tag sequence
may adopt a native-like conformation without disrupting global folding of the helical target. Additionally,
the proteins have similar or marginally increased thermal stabilities and demonstrate cooperative,
reversible unfolding. Expanding on this, we show that a similar 17-amino acid hairpin sequence can also
serve as an expression-enhancing tag when fused to either terminus of the same poorly-expressed helical
proteins, and we are currently investigating possible mechanisms for this finding.
POST 12-182
Gradient Diversity Enriches Combinatorial Protein Library Design
Daniel R. Woldring, Benjamin J. Hackel
Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, US
Discovering new protein functionality via combinatorial library currently lacks efficiency, particularly in
small ligand scaffolds. This study hypothesizes that combinatorial protein libraries would benefit from
optimizing site specific amino acid distribution rather than the traditional heavily diversified paratope and
fully conserved framework. The latter methodology is vulnerable to overly destabilizing the paratope as
well as missing beneficial intramolecular contacts with the new paratope. We examined the hypothesis
that the most efficient evolution is achieved with a combinatorial library exhibiting a gradient of diversity
from extensive diversity in the paratope hot spot to full conservation in the framework. Importantly, this
gradient includes moderate diversity, with structural bias, within the paratope interfacing with target yet
peripheral to the hot spot. Moreover, more mild diversity is included adjacent to the interfacial residues to
yield optimal intramolecular contacts with the newly identified paratope. To test the hypotheses, we
investigate the evolvability of small proteins in the context of binding affinity using the beta-sheet
140
POSTER ABSTRACTS
fibronectin(III) and the three-helix affibody domain. Naïve combinatorial libraries were constructed to
incorporate gradient diversities across individual sites that were hypothesized to frequently be involved
with protein-protein interactions or of structural importance. Yeast surface display and magnetic- and
fluorescence-activated cell sorting were used to isolate clones demonstrating high affinity, specific
binding to six protein targets. Thousands of unique and highly functional clones were isolated, sequenced
via Illumina MiSeq and analyzed to elucidate the most evolutionarily favorable amino acid distributions
within 25 fibronectin sites and 15 affibody sites. We found that a subset of fibronectin sites demonstrate
improved evolvability by implementing only mild diversity, with a wild-type bias, such as T76, a semiburied position in the peripheral paratope. Additionally, restricting diversity at structurally relevant
positions – both within (V29 and G79) and distal to (D23 and S85) the paratope – is evolutionarily
favorable. Collectively, our results provide an efficient strategy for quantifying the balance between interand intra-molecular interaction potential at each scaffold position to optimize amino acid distributions for
enhanced combinatorial library designs.
POST 12-183
Expanding The GFP Toolbox Through A Better Understanding Of Its Folding Pathway
Keith Fraser1, 2, Colleen Lamberson1, 2, Victoria Jones1, 2, Erin Gilbert1, 2, David Rosenman1, 2, Yao-ming
Huang3, Derek Pitman4, Shounak Banerjee1, 2, Yan Xia5, Angela Choi1, 2, Rachel Altshuler1, 2, Luis Garreta6,
John Karonicolas5, Donna E. Crone1, Jonathan S. Dordick1, 2, 7, Christopher Bystroff1, 2, 8
1
Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 2Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, US, 3UCSF, San Francisco,
California, US, 4Institute of Genomics and Proteomics, UCLA, Los Angeles, California, US, 5Molecular
Biosciences, University of Kansas, Lawrence, Kansas, US, 6Universidad del Valle, Cali,
Colombia, 7Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York,
US, 8Computer Science, Rensselaer Polytechnic Institute, Troy, New York, US
During the biotech explosion over the last 20 years we have seen an ever expanding role for the green
fluorescent protein (GFP) in vivo and in vitro assays. One of the primary drives of this has been the work
done to broaden the spectroscopic range of this protein. These successes have highlighted how amenable
GFP is to design. With this in mind we attempted to take advantage of the designability of GFP in an
attempt to improve its folding efficiency and better understand its folding pathway.
Wt GFP has been reported to have a tendency to misfold in vitro; in an attempt to fix this problem we
targeted the cis proline at P89 in GFP and redesigned this position and some surrounding residues. The
engineered mutant (all trans GFP or AT-GFP) showed faster folding kinetics and no misfolding. We have
observed that the introduction of a cis peptide bond (G57P58) significantly impedes folding. We were able
to improve the folding efficiency of this mutant on the order of the wt via a G57A point mutation.
We have previously reported that GFP tolerates the omission of one of its secondary structural elements
(LOO-GFPs). Depending on the structural element that is removed the ability of the rest of the molecule
to fold and form a soluble protein varies. Co-expression with the missing element results in reconstitution
of fluorescence to varying degrees. We interpret preservation of native like structure in LOO-GFPs as an
indicator of the order of folding of secondary structural elements. CD data along with equilibrium and
stop-flow fluorescence data have allowed us to further refine our folding pathway model for GFP. We
have also supplemented these experimental data with a computational model of the intermediates
present along the GFP unfolding pathway.
POST 12-184
Are Peptide Nucleic Acids (PNAs) Recognized by Aminoacyl-tRNA Synthetases (aaRSs)?
Crystal Serrano1, Long Nguyen1, Anthony Bell1, Filbert Totsingan2
141
POSTER ABSTRACTS
1
University of Southern Mississippi, Hattiesburg, Mississippi, US, 2New York University, New York, New
York, US
PNAs (peptide nucleic acids) are synthetic hybrids of nucleic acids and peptides. PNA was originally
discovered during the development of gene targeting drugs. Like DNA and RNA, PNA possess standard
purine and pyrimidine bases. As do peptides and proteins, PNA contains a polyamide backbone –
typically aminoethyl glycine (AEG). The neutral PNA backbone provides greater stability than the natural
ribose-phosphate in hybridization to complimentary strands of DNA and RNA. Hence, it is not surprising
that PNA has been investigated extensively as an antigene reagent. The seminal report by Peter Nielsen
and Orgel clearly showed that PNA is capable of transferring genetic information to RNA via templatedirected syntheses. These data indicate that PNA might have played a key role in the establishment of
primordial protein/peptide translation systems. The objective of this study was to determine if PNAs are
recognized by extant translational machinery such as aminoacyl-tRNA synthetases (aaRSs). Here, we
32
show, using a P based aminoacylation assay, that PNA-RNA duplexes are efficiently recognized and
aminoacylated by AlaRS. The relative charging efficiency of PNA-RNA duplexes are comparable to RNAAla
RNA duplexes based on tRNA mini- and micro-helices. The biophysical characteristics of PNA-RNA and
RNA-RNA duplexes were evaluated using: electrophoretic mobility shift assays (EMSA), circular dichroism
(CD) and UV spectroscopy. The CD investigations revealed that, as expected, RNA-PNA duplexes were
o
more stable (D Tm ≈ 10 C) than the corresponding RNA-RNA duplex. EMSA and UV analysis showed that
PNA-RNA duplexes form more readily at lower pH values. This is the first investigation that shows aaRSs
charge synthetic nucleic acids (PNA). Future investigations will focus on screening PNAs against aaRSs to
develop antibiotics.
POST 12-185
Protein Rings and Tubes as Versatile Templates for Self-Assembled Bionano Structures
Ali A. Malay2, Zuben Brown1, Kenji Iwasaki1, Jonathan G. Heddle2
1
Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, and
CREST, Japan Science and Technology Agency, Osaka, Osaka, Japan, 2Heddle IRU, RIKEN, Wako, Saitama,
Japan
The natural world demonstrates the power of proteins to act as self-assembled nanometric structures able
to carry out a diverse range of interactions with matter including as nanostructural components, catalysts
and nanomachines. It would therefore be very useful to build our own proteins to carry out reactions and
functions that we desire. Unfortunately current technology does not allow us to design proteins of
significant size ab initio. An approach to overcome this limitation is to use naturally occurring proteins
and alter their properties such that they assemble into the desired structures with desired properties (a
semi-synthetic approach). In this work we will summarize our progress with two proteins, the nanotubular
tomato mosaic virus (ToMV) and the toroidal protein TRAP (trp-RNA binding attenuation protein). These
proteins have proved very useful as templates for the production of non-biological nanotubes and
nanowires with potential applications in electronics and sensors and as building blocks for self-assembly
of virus-like particles via unique catalytic chemistries.
POST 12-186
Bio-Inspired Tectons: The Architecture & Engineering Of Synthetic Ring-Forming Proteins
Francesca Manea, Bridget C. Mabbutt
Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, New South
Wales, Australia
142
POSTER ABSTRACTS
The vast structural and chemical diversity of proteins makes them highly attractive as bio-building blocks
or ‘tectons’ for fabrication of materials by self-assembly. Through supramolecular engineering, quaternary
structure and function can be manipulated to fabricate materials with new or improved properties. I am
exploiting self-assembling Lsm proteins to construct novel nanostructures of ring morphology. In
vivo, the Lsm proteins form the core of the ribonucleoprotein (RNP) complexes crucial to RNA
metabolism. They assemble as oligomeric rings via the association of seven distinct Lsm protomers to
generate an RNA-binding scaffold. I have engineered synthetic Lsm rings composed of fused protein
components, so creating simplified structures with reduced symmetry. In solution, recombinant Lsm selforganise into overlaid ring pairs through electrostatic interactions mediated by salt concentration. I have
further driven oligomerisation of Lsm ring tectons into coherent architectures by engineering Cys residues
into ring faces for covalent stacking. These disulfide-bonded Lsm clusters are readily disengaged by a
reducing step. The cluster architecture of high MW Lsm complexes has been visualised by electron
microscopy (EM) to augment solution-state studies. Crystallisation of Lsm forms offers potential to
understand the quaternary forms, from clusters to tubules. Other avenues of conjugation between Lsm
rings are also being explored, including self-assembling peptides and metal-binding via engineered Histags. Ultimately, these novel nanomaterials will have applications in RNA housing and delivery capsules or
as next-generation bio-inspired nanosensors.
POST 12-187
A Designed Small Protein For Controlling Site-Specific Mineralization Of Silica And Calcium On
Dnas
Kenji Usui2, Kazuma Nagai2, Hiroto Nishiyama2, Aoi Yamada2, Makoto Ozaki2, Takaaki Tsuruoka2, Kin-ya
Tomizaki1 1Department of Materials Chemistry, Ryukoku University, Otsu, Japan, 2Faculty of Frontiers of
Innovative Research in Science and Technology, Konan University, Kobe, Japan
Biomineralization, (mineralization in a biological system), precipitation of inorganic compounds, is
controlled by some natural proteins. These proteins can control spatiotemporally both the size and the
shape of the inorganic deposits with high reproducibility and accuracy during the biomineralization
processes. Short peptides derived from sequences of these biomineralization proteins can also precipitate
inorganic compounds [1]. Because these peptides are easy to design and synthesize compared to the
natural proteins, they are promising molecules for the construction of organic-inorganic nanostructures
with mineralization. There have been few studies to date using these peptides for well-controlled sitespecific mineralization yielding homogeneously-shaped inorganic precipitates.
In this study, we
focused on silica and calcium mineralization and demonstrated a site-specific mineralization using DNA
and designed peptides with peptide nucleic acids (PNA) [2]. After design and synthesis of the peptides,
we checked its ability of silica mineralization using atomic force microscopy (AFM), dynamic light
scattering (DLS), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy
(EDX). Then we attempted more complicated mineralization. Calcium as the second mineralized
inorganic compound, was precipitated on the silica-peptide-DNA structure. Preliminarily, TEM-EDX
images showed dumbbell shape nanostructures, long chains for calcium-DNA and nanometer-sized
spheres for silica and calcium precipitates.
Our results will provide good examples of the controlled
distribution of inorganic compounds and organic compounds. Such system would be one of the most
powerful tools for bottom-up methods in the nanobiotechnology and material fields.
[1] Knecht, M.R.;
Wright, D.W. Chem. Commun. 2003, 3038. [2] Sano, S.; Tomizaki, K.-Y.; Usui, K.; Mihara, H. Bioorg. Med.
Chem. Lett. 2006, 16, 503.
POST 12-188
Enhancing Predictions Of Surface Entropy Reduction For Improved Crystallizability Of Proteins
143
POSTER ABSTRACTS
Derek J. Pitman1, Thomas Holton1, Luki Goldschmidt2, 1, Zygmunt Derewenda3, David Eisenberg1
1
University of California, Los Angeles, Los Angeles, California, US, 2University of Washington, Seattle,
Washington, US, 3University of Virginia, Charlottesville, Virginia, US
The resistance of certain proteins to forming well-ordered crystals impedes the determination of highresolution structures and expends considerable resources on unsuccessful crystallization trials. A
successful strategy for improving the crystallizability of these problematic proteins is surface entropy
reduction (SER), the mutation of high-entropy residues such as lysine or glutamate on the protein surface
to low-entropy residues such as alanine, facilitating the formation of crystal contacts. We have applied
this concept to develop a prediction server, SERp, which takes a primary sequence as input and uses
expected surface entropy, secondary structure prediction and evolutionary conservation to determine
probable sites for SER mutations (Goldschmidt et al., Protein Science 16(8):1569-76). The SERp server has
allowed for the determination of numerous new crystal structures. SERp can also interface with the
XtalPred server for prediction of protein crystallizability (Slabinski et al., Bioinformatics 23(24):3403-5).
Here we review the SERp server in its current form, outline a future revision of the server infrastructure,
and discuss potential extensions for the methodology, including improved predictions for membrane
proteins and an increased emphasis on the use of known structural data and previous SER successes in
refining the entropy reduction theory. Entropy reduction can thus be viewed as a mechanism to explore
the more general design of crystal contacts. The SERp server can be accessed at:
http://services.mbi.ucla.edu/SER/
POST 12-189
Repeat Protein Scaffolds For Assembly Of Functional Nanostructures
Sara H. Mejias1, 2, Pierre Couleaud1, 2, Javier Lopez3, 4, Begoña Sot1, 2, Carmen Atienza3, Teresa
Gonzalez4, Aitziber L. Cortajarena1, 2
1
Nanobiosystems, IMDEA Nanociencia, Madrid, Spain, 2CNB-CSIC-IMDEA Nanociencia Associated Unit,
Madrid, Spain, 3Química Orgánica, Universidad Complutense, Madrid, Spain, 4IMDEA Nanociencia,
Madrid, Spain
Self-assembly of biological molecules into defined functional structures has a tremendous potential in
nanopatterning, and the design of novel materials and functional devices. Molecular self-assembly is a
process by which complex three-dimensional structures with specified functions are constructed from
simple molecular building blocks. We present the study and characterization of the assembly properties of
modular repeat proteins, in particular designed consensus tetratricopeptide repeats (CTPRs) which
1
stability and function can be tuned, and their appplication as building blocks in order to generate
2
functional nanostructures and materials.
1. We show the assembly of repeat proteins into thin
nanometric fibers. We have characterized the polymerization kinetics and thermodynamics and developed
a quantitative simple model of the polymerization for rational control of features of the final
3
nanostructures (Fig. 1a). 2. We show data on the design and conjugation of CTPR building blocks with
gold nanoparticles (Fig 1b) and how these protein scaffolds are used for nanometer-precise arrangement
of the nanoparticles, toward the generation of nanoscale systems with numerous appplications in
nanotechnology. 3. We show preliminary data on the use of CTPR proteins to template donor-acceptor
pairs for electroactive materials. In order to achieve an efficient electron transfer the arrays of molecules
need to be ordered with defined inter-molecular distances. We show the conjugation of CTPRs scaffolds
with electroactive molecules and the characterization of the hybrid structures (Fig. 1c). References 1. (a)
Cortajarena, A. L.; Regan, L., Calorimetric study of a series of designed repeat proteins: modular structure
and modular folding. Protein Sci 2011, 20, 341-352; (b) Cortajarena, A. L.; Liu, T. Y.; Hochstrasser, M.;
Regan, L., Designed proteins to modulate cellular networks. ACS Chem. Biol. 2010, 5 (6), 545-552.
2. Mejias, S. H.; Sot, B.; Guantes, R.; Cortajarena, A. L., Controlled nanometric fibers of self-assembled
144
POSTER ABSTRACTS
designed protein scaffolds. Nanoscale 2014. 3. Grove, T. Z.; Regan, L.; Cortajarena, A. L., Nanostructured
functional films from engineered repeat proteins. J. R. Soc. Interface. 2013, 10(83), 20130051.
POST 12-190
Generation of Novel Amino Acid Dehydrogenase Activity through Evolution of a Highly Specific
Alanine Dehydrogenase
Emily Mundorff
Chemistry, Hofstra University, Hempstead, New York, US
Mutants of alanine dehydrogenase (AlaDH) from the M. tuberculosis have been identified with activity
towards leucine, norleucine and norvaline. This enzyme shares no significant structural or sequence
similarity with the Phe/Glu/Val/Leu Dehydrogenase superfamily and has been demonstrated to have high
specificity towards alanine. Position F94 was identified through molecular modeling as being essential
for substrate specificity. Modeling studies predicted that mutating this residue to either alanine or serine
would create room in the binding pocket for bulkier substrates. Indeed both mutants facilitated the
generation of activity on leucine, norleucine and norvaline while decreasing the activity for its natural
substrate. Saturation mutagenesis of the active site identified improvements upon these activities. This
result indicates that even though this enzyme bears no significant relationship to other amino acid
dehydrogenases, it still has the potential to evolve these functions.
POST 12-191
Rational Protein Design, SPR Binding Analysis, And Iterative Optimization Of Student-Designed
Single-Chain Immunoreceptors
Benjamin J. McFarland, Andrew Daman
Chemistry and Biochemistry, Seattle Pacific University, Seattle, Washington, US
The success of rational protein design can be enhanced by synthesizing and testing multiple designed
proteins in the lab. We previously optimized binding of the MICA protein surface to its protein ligand,
the homodimeric NKG2D immunoreceptor, with an iterative process resulting in seven MICA mutations
that successfully enhanced the interaction. Here we apply these techniques to designing a single-chain
form of NKG2D with an optimized homodimer interface. To effectively assess the binding of multiple
designs, we assign multiple undergraduates to make the proteins in a biochemistry teaching lab course
and to test them for binding in a survey of physical chemistry lab course, guiding and recording their
progress with a set of GENI online tools we have developed. Surface Plasmon Resonance (SPR) binding
analysis was made possible in this context with a BIAcore X instrument, which gave similar results as a
BIAcore T100 instrument at <10% of the cost. Six linker designs were produced, purified, and tested,
ranging from traditional gly-ser linkers to structure-based designs. The structure-based designs resulted
in higher protein-protein affinities. Rosetta was used to redesign the NKG2D homodimer interface in
145
POSTER ABSTRACTS
two areas: by replacing a disulfide bond network at the top of the interface near the introduced linker,
and in the hydrophobic center of the interface. Single-chain NKG2D versions with structure-based linkers
containing optimized residues in each of these areas and in both areas together were produced and
tested for binding. The redesigned NKG2D with the highest affinity will be used as the basis for future
designs, such as optimization of the NKG2D interaction with MICA and studies of NKG2D-ligand
specificity. [Partial support for this work was provided by the National Science Foundation's
Transforming Undergraduate Education in Science, Technology, Engineering and Mathematics (TUES)
program under Award No. 1322848.]
POST 12-192
Efficient Gene Disruption At Htert Promoter Region By Simultaneous Digestion By Pairs Of ZFNs Or
Guide RNAs For CRISPR/CAS System
Wataru Nomura, Akemi Masuda, Hirokazu Tamamura
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan
Artificial zinc finger proteins (ZFPs) consist of Cys2-His2-type modules composed of about 30 amino acids
with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can
substitute for the binding domains of enzymes that act on DNA to create designer enzymes, such as
nucleases, recombinases [1], and methylases [2], with programmable sequence specificity. Genome editing
and modification by the enzymes could be applied for many fields of basic research and medicine. Recent
findings of new platforms for genome editing such as transcriptional activator-like effector (TALE) and
CRISPR/Cas system expands the possibility of the technologies. These technologies enable efficient
knock-out of gene of interest or regulation of gene function. In this study, ZFN pairs targeting promoter
region of human telomerase reverse transcriptase (hTERT) were designed and constructed. Endogenous
gene targeting by the paired ZFN showed successful induction of mutation at the upstream- and
downstream-site of core promoter region of hTERT. The simultaneous digestion by both pairs of ZFNs
showed enhanced disruption of the target gene. Guide RNAs for CRISPR/Cas9 system targeting the same
sequences with ZFN were also constructed and tested. The enhancement of gene disruption by the
simultaneous digestion at the upstream- and downstream-sites was observed as shown in the ZFN
experiments. Cells with gene disruption at the core promoter region of hTERT showed decreased
expression of hTERT. The study will provide the information for the role of hTERT promoter activity
expressing cell-specific phenotypes such as immortality of cancer cells. Furthermore, the efficient genome
editing at the specific promoter region could be applied for studies in Synthetic Biology. [1] Nomura, W.,
Masuda, A., Ohba, K., Urabe, A., Ito, N., Ryo, A., Yamamoto, N., Tamamura, H. Biochemistry, (2012) 51,
1510-1517. [2] Nomura, W., Barbas, C. F., III J. Am. Chem. Soc., (2007) 129, 8676-8677.
POST 12-193
High-Throughput Sorting Of The Highest Producing Cell Via A Transiently Protein-Anchored
System
Kuo-Hsiang Chuang
Taipei Medical University, Taipei, Taiwan
Developing a high-throughput method for the effecient selection of the highest producing cell is very
important for the production of recombinant protein drugs. Here, we developed a novel transiently
protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection
of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial
growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse
B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored
form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of
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POSTER ABSTRACTS
secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a
2
high correlation (R =0.9165) between the secretion level and surface expression levels of αEGFR Ab. These
results suggested that the novel transiently protein-anchored system can easily and efficiently select the
highest producing cells, reducing the cost for the production of biopharmaceuticals.
POST 12-194
Dissecting Contributions To Protein Stability Via Recombination Of A Wild Type And
Computationally Designed Protein
Lucas Johnson1, Lucas Gintner1, Luke Minardi2, Christopher Snow1
1
Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado, US, 2Rowan
University, Glassboro, New Jersey, US
Despite attempts to improve accuracy and reliability in computational protein design, progress is often
hindered by the binary success or failure outcome inherent in the design method. Expanding from a single
test sequence to a library of diverse designs has the potential to improve understanding and facilitate an
iterative learning process. Isolating regions that positively or negatively impact protein fitness can also
help focus analysis and accelerate subsequent design steps. In this work, we demonstrate how sitedirected chimeragenesis via SCHEMA recombination (Voigt et al. 2002) can be used to generate a
computational design library. The methods are applied to a novel test case, Endoglucanase E1
from Acidothermus cellulolyticus. Initially, consensus sequence analysis and Fold-X energy predictions are
combined to generate a candidate design space, and a single optimized design is identified based on the
Rosetta energy function in the SHARPEN design suite (Loksha et al.2009). Recombination with the wild
type sequence then creates a test library of sixteen chimeras. By characterizing the thermal stability and
activity of each chimera, neutral and destabilizing regions can be decomposed using linear regression.
Ongoing molecular dynamics analysis focuses on the destabilized regions in an attempt to further
elucidate design deficiencies.
Loksha, I. V., Maiolo, J. R., Hong, C. W., Ng, A. & Snow, C. D. Journal of
Computational Chemistry 30, 999–1005 (2009). Voigt, C. A., Martinez, C., Wang, Z.-G., Mayo, S. L. &
Arnold, F. H. Nat. Struct. Biol. 9, 553–558 (2002).
POST 12-195
Computational Design of Protein Ligand Interfaces Using RosettaLigand
Brittany Allison, Jens Meiler
Chemistry, Vanderbilt University, Nashville, Tennessee, US
The computational design of proteins that bind small molecules is one of the unsolved challenges in
protein engineering. The relatively small size of the ligand limits the number of intermolecular
interactions, and near perfect geometries between interacting partners are required to achieve high
binding affinities. For apolar, rigid small molecules the interactions are dominated by short-range van der
Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cationπ, and π-π interactions gain importance. The ability to computationally recapture and predict native-like
interactions with high accuracy and efficiency would be an asset in biotechnology and medicine, such as
for therapeutic development, enzyme design, and engineering functional proteins. To assess the current
state of protein-ligand interface design, we benchmarked the computer algorithm Rosetta on a diverse
set of 43 protein-ligand complexes. On average, we achieve sequence recoveries in the binding site of
59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full
reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues
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POSTER ABSTRACTS
that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full
ligand reorientation was allowed.
POST 12-196
Eliminating Endotoxin at the Source -A Novel Competent Cell Line with Modified
Lipopolysaccharide for Low-Endotoxin Plasmid Production
Saurabh Sen1, Uwe Mamat2, Chad Souvignier3, Eric Steinmetz1, Chelsea Kovacich1, David Mead1, Curtis
Knox1
1
Lucigen Corporation, Middleton, Wisconsin, US, 2Research Center Borstel, Borstel, Germany, 3 Research
Corporation Technologies, tuscon, Arizona, US
The Gram-negative bacterium E. coli is the workhorse of molecular biology, regularly used as the primary
host for DNA cloning and protein production. One of the major limitations in using E. coli relates to the
lipopolysaccharide (LPS) component of the outer membrane. LPS is an endotoxin that is a potent activator
of many immune cells through the Toll-like receptor-4 (TLR4) and can directly trigger endotoxic shock
(septic shock), resulting in severe medical problems and death. In plasmid production applications, the
presence of LPS is widely accepted to have a negative effect on mammalian cell transfection efficiencies,
cell viability, and downstream protein production. Such a toxic component requires expensive and time
consuming purification to remove it. Current methods for endotoxin-free plasmid prep are varied, but in
general cost ten times as much with lower yields than standard plasmid prep kits. Eliminating LPS from
the E. coli outer membrane is a novel approach for producing plasmid DNA for mammalian transfection
without the need for expensive endotoxin-free plasmid cleanup kits. Lucigen, in combination with
Research Corporation Technologies, has developed a new line of E. coli competent cells called
ClearColi™. These cells have been genetically modified to remove the immune response triggers
associated with LPS while still retaining viability and plasmid production capabilities. By eliminating the
offending LPS, purification of plasmid DNA for downstream applications is reduced to simpler, less
expensive processes. Transfection efficiencies and protein expression levels in mammalian cells are equal
to or greater than from plasmids prepared via endotoxin-free purification kits.
POST 12-197
Computational Design of Leucine-Rich Repeat- based Protein Binding Scaffolds.
Sebastian Rämisch2, Ulrich Weininger1, Ingemar André2
1
Biophysical Chemistry, Lund University, Lund, Sweden, 2Biochemistry and Structural Biology, Lund
University, Lund, Sweden
Repeat protein- based scaffolds for the development of protein binders have a number of advantages
over antibodies. Several consensus designs of all-alpha repeats, e.g. DARPins and CTPRs, are already in
use as platforms to introduce novel binding functionalities. However, using scaffolds based on highly
conserved core-residues leaves the huge diversity of natural repeat protein geometries entirly unutilized.
Proteins comprised of leucine-rich repeats (LRRs), which have a characteristic extented beta-sheet, show a
particularly high diversity in terms of curvature and helical twist. We wanted to test, whether
computational de novo design can be used to design an LRR-protein with an artificial geometry in a
highly controlled manner. Using Rosetta for fixed-backbone sequence optimization and de novo
backbone design, we were able to obtain sequences that can be highly overexpressed in E.coli. Extensive
biophysical characterization showed, that only constructs containing conserved cysteines are correctly
folded and stable at high concentrations and elevated temperatures. In contrast to affinity maturation
starting from a fixed scaffold, our approach enables the design of LRR- based scaffolds where not only
surface residues, but also the overall shape of a scaffold can be adapted for optimal binding of target
molecules.
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POSTER ABSTRACTS
POST 12-198
Generation and Characterization of the Native-like IgG Bispecific Antibodies
Xiufeng Wu1, Steven M. Lewis2, Arlene Sereno1, Flora Huang1, Anna Pustilnik1 , Heather L. Rick1, Elaine
M. Conner1, Shane Atwell1, Brian Kuhlman2, Stephen J. Demarest1
1Eli Lilly and Co, San Diego, CA; 2Department of Biochemistry and Biophysics, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599
Bispecific antibodies (BsAbs), which target two antigens or epitopes and integrate the activity of two
separate monoclonal antibody (MAb) therapeutics, represent a future direction for achieving more
meaningful and durable therapeutic responses in various diseases. However, robust generation of fully
IgG bispecific antibodies has been a premier challenge in the field of antibody and protein engineering
for decades. Existing methodologies for generating these molecules have required extensive engineering
of every molecule, discovery of antibodies using common light chains, or biochemical processing post
expression – and most alter the native antibody geometry to achieve bispecific binding. Here, we describe
the computational and rational design of an orthogonal antibody heavy chain and light chain interface
that facilitates the simultaneous co-expression of two Fab moieties with improved assembly of correct
heavy chain/light chain pairs. Utilizing this novel Fab interface, we demonstrated the ability to express two
antibody HCs and LCs and have them assemble with improved specificity into native-like IgG
BsAbs. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable
properties of native IgG. As such, these bispecific reagents may be useful in many biotechnological
applications.
POST 12-199
Improving The Efficiency Of Concanavalin A as Affinity Ligand in Alkaline ph Range
Akash Chaudhary1, Shakeel Ahmad1, Shadab Ahmad2, Mohd. Tashfeen Ashraf1
1
School of Biotechnology, Gautam Buddha University, Greater Noida, India, 2School of Life Sciences,
Jawaharlal Nehru University, New Delhi, Delhi, India
The lectin Concanavalin A (Con A) is a homotetramer that requires metal ions for its binding to sugars like
glucose and mannose. It is also referred to as a dimer of dimers as the native structure is the result of
amalgamation of two homodimers. Con A is an important affinity ligand that is routinely used for
purification of glucose/mannose containing glycoproteins. However, Con-A bound matrix is rarely
effective beyond pH 9 as the lectin has great tendency to form higher aggregates beyond pH 7. Our study
aims to identify the residues/regions crucial for this increased tendency of Con A to form aggregates in
alkaline pH range. The studies performed so far points towards the involvement of specific amino acid
residues, the presence of which may probably be making these regions sticky. Identifying such sticky
residues/regions may eventually help in designing the Con A that has reduced tendency to from
aggregates as compared to its wild type counterpart.
POST 12-200
Protein-Based Stimuli-Responsive Hydrogels For Targeted Drug Delivery
Ashley C. Schloss1, Abbie Omolu2, Richard Day 2, Lynne J. Regan1, 3
1
Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, US, 2Applied
Biomedical Engineering, University College London, London, United Kingdom, 3Chemistry, Yale
University, New Haven, Connecticut, US
Novel materials whose properties respond to external stimuli have a variety of important applications.
Proteins are the cell’s machines, and perform innumerable functions in response to chemical and physical
149
POSTER ABSTRACTS
signals. A natural way to incorporate stimuli-responsiveness into materials, therefore, is to build them
from proteins. Creating hydrogels from such components has the additional advantage that proteins are
mono-disperse, thus avoiding issues related to typically used, non-homogeneous components such as
PEG and PGLA. Moreover, functionalization of proteins can be efficient and site-specific, allowing a vast
array of additional applications. Until recently however, the use of solely proteins as smart material
components has been limited by their underlying linear construction and lack of branching. The
introduction of covalent branches would greatly increase the properties and potential of using proteins in
materials research. Here we present the design and characterization of materials in which we use
isopeptide bond formation between the peptide ‘Spytag’ and its cognate protein ‘Spycatcher’ to create
novel, covalently branched proteins. The designs we present greatly extend the recently described
technology (W. Zhang, et. al. J. Am. Chem. Soc., 2013)
POST 12-201
The β/α and α/β of TIM Barrel Proteins: Database and Structural Analysis
Ramakrishna Vadrevu, Rajashekar V. Kadumoori
Biological Sciences, Birla Institute Of Technology & Sciences, Hyderabad, Andhra Pradesh, India
The (βα)8 / TIM barrel is one of the most common folds of known protein structures facilitating a wide
variety of catalytic functions. The fold is formed by the repetition of the basic βαβ building block in which
the β-strands are followed by α-helices eight times alternating in sequence and structure. The pair of
adjacent parallel β-strands and the intervening anti-parallel α-helix, i.e., the βαβ module, serve as the
minimal unit of stability. The intervening β/α and α/β loops that connect β-strands to the α-helices and
the α-helices to the β-strands play an important role in catalysis and stability respectively. Given the
versatility and the segregation of the role of loops in function and stability, TIM barrels proteins could
serve as excellent candidates for protein engineering through the strategy of swapping loops between
different proteins without compromising functionality and conformational stability of target proteins. The
availability of a large number of TIM barrel protein structures in Protein Data Bank has motivated us to
perform a detailed and dedicated analysis of the β/α and α/β loops from TIM barrel proteins with a view
to develop a repository of structurally similar loops for loop swapping between TIM barrel proteins but
also to identify potential loops to help design stable bab folding motifs. From ~250 non redundant TIM
barrels bearing sequence homology of <30%, a total of 3628 loops were extracted, grouped as 1742 and
1886 α/β loops and β/α loops respectively, and analyzed for their amino acid propensities, loop size, turn
conformation types etc. A database of TIM barrel loops has been developed along with a web based
Graphical User Interface to superimpose and identify compatible loops. This may help in modeling loop
swapping between different TIM barrel proteins.
POST 12-202
De Novo Design of Peptides that Assemble Lipid Nanodiscs&rlm;
Shao-Qing Zhang2, 1, Kazuma Yasuhara2, 3, Hyunil Jo2, William F. DeGrado2
1
University of Pennsylvania, Philadelphia, Pennsylvania, US, 2University of California at San Francisco,
San Francisco, California, US, 3Nara Institute of Science and Technology, Ikoma, Nara, Japan
A native lipid bilayor environment is very important for studying the structure and function of integral
membrane proteins. Certain amphiphilic peptides wrap lipid molecules to assemble water-soluble,
homogeneously sized discoidal nanoparticles (termed nanodiscs) into which membrane proteins can be
reconstituted. De novo design of different amphiphilic peptides that form nanodiscs of various sizes is a
challenging job, as peptidic amphiphilicity itself can generate complex self-assembly behaviours. We
employ a sequence-based strategy to design a series of 22-residue-long nanodisc-forming alpha-helical
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POSTER ABSTRACTS
peptides that assemble with lipid molecules in a "picket-rail" orientation (where the peptides are parallel
to the lipid molecules). The formation of nanodiscs is confirmed by electronic microscopy. Dynamic light
scattering experiments show that the peptides form nanodiscs with a monodisperse particle size
distribution of a diameter ranging from 15 to 20 nm. Strong enhancement in helical secondary structure is
observed by circular dichroism when the peptides are titrated with lipid components. Notably, NMR
analysis shows that the nanodiscs can tumble freely in solution, which makes it amenable to structural and
functional studies of reconstituted membrane proteins by solution NMR. Consistent with the design,
modulation of the sequence at the peptide interaction interface leads to a drastic change in
concentration-dependent assembly behaviour. The design of nanodisc-forming peptides opens new
avenues for customizing membrane protein reconstitution systems by size, and also for studying the selfassembly behaviours of amphiphilic peptides to wrap around lipid molecules.
POST 12-203
A Hyperstable Minimalist Protein for Molecular Recognition
Michael W. Traxlmayr1, 4, Raja R. Srinivas2, Elisabeth Lobner3, Bruce Tidor2, Karl D. Wittrup1, 2, 4
1
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts, US, 2Department of Biological Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts, US,3University of Natural Resources and Life Sciences, Vienna, Austria,
Austria, 4Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, Massachusetts, US
Antibodies are the most rapidly growing class of therapeutic proteins. Their most important properties are
their high affinity and specificity for a given target molecule. In recent years there has been increasing
interest in alternative binder scaffolds, which combine high affinity and specificity with other desired
properties, such as high stability and solubility, as well as low propensity for aggregation and small size.
Although significant progress has been made in the development of alternative binder scaffolds during
the last years, there is still room for improvement. One promising candidate is the DNA-binding protein
Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus. This protein combines the
advantages of high stability (Tm value of 98°C), high solubility, lack of cysteines and small size (7 kDa). In
recent studies the potential of Sso7d for the construction of binders against various target molecules has
been demonstrated. However, since Sso7d is a DNA-binding protein, it contains a high number of positive
charges, with 22% of all amino acids being lysines. Here, we show that the positively charged residues
cause unspecific binding to mammalian cells. Importantly, we also demonstrate that this stickiness can be
completely eliminated by reducing the number of positive charges. In fact, a direct correlation between
the net formal charge of the protein and unspecific binding to mammalian cells was observed. Currently
yeast surface display libraries based on the reduced charge-Sso7d scaffold are constructed. We anticipate
that those libraries will yield binders with high affinity, stability and specificity for a variety of
applications.
POST 12-204
A Synthetic Biochemistry Molecular Purge Valve Module that Maintains Redox Balance
Paul Opgenorth
Biochemistry, UCLA, Los angeles, California, US
The greatest potential environmental benefit of metabolic engineering would be the production of high
volume commodity chemicals, such as biofuels. Yet the high yields required for the economic viability of
low-value chemicals is particularly hard to achieve in microbes due to the myriad competing biochemical
pathways. An alternative approach, which we call synthetic biochemistry, is to eliminate the organism by
constructing biochemical pathways in vitro. Viable synthetic biochemistry, however, will require simple
151
POSTER ABSTRACTS
methods to replace the cellular circuitry that maintains cofactor balance. Here we design a simple purge
+
valve module for maintaining NADP /NADPH balance. We test the purge valve in the production of
polyhydroxybutyryl bioplastic and isoprene – pathways where cofactor generation and utilization are
unbalanced. We find that the regulatory system is highly robust to variations in cofactor levels and readily
transportable. The molecular purge valve provides a step toward developing continuously operating,
sustainable synthetic biochemistry systems.
POST 12-205
“PA Tag”, A Versatile Affinity Tag System That Enables One-Step Affinity Purification And High
Sensitive Detection Of Recombinant Proteins From Dilute Sample
Yuki Fujii
Institute for Protein Research, Osaka, Japan
A rat monoclonal antibody NZ-1 recognizes PLAG domain sequence of human podoplanin with a very
high affinity. Recently, we developed a novel tag purification and detection system using NZ-1 and the
epitope peptide dubbed PA tag [1]. PA tag consists of 12 amino acid residues (GVAMPGAEDDVV), and
requires a minimum length of 10 residues to be recognized by NZ-1. Alanine scanning mutagenesis
revealed that two single amino acid mutations (M4A and D10A) would completely destroy the binding,
indicating the major contribution of these residues in the recognition. We analyzed the binding affinity of
NZ-1 toward PA tagged protein, using T4 lysozyme as a model protein. Fusion of PA tag at N- or Cterminal of T4L resulted in very high binding affinity with KD values of ~10-9 M or ~10-12 M, respectively.
The high affinity was ensured by extremely slow dissociation rate. Nevertheless, it was possible to
dissociate bound antigen from the antibody by incubating with free epitope peptide at 0.1 mg/ml. The
very high affinity and specificity also enabled detection of PA-tagged proteins in the Western blot format
with very short incubation time (<5 min) with virtually no nonspecific bands. Furthermore, the PA-tagged
proteins expressed on cell surface could be detected by flow cytometry after staining with NZ-1 at
concentration as low as 0.1 μg/ml. Using NZ-1-immobilized Sepharose resin, we could rapidly purify
various recombinant glycoproteins attached with PA tag from dilute cell culture supernatant in one step.
The regeneration of NZ-1 resin can be accomplished by washing with non-denaturing buffer (3 M MgCl2,
pH 6.0), which enabled us to reuse the affinity column over 60 times. Finally, we have determined the Xray crystal structure of the NZ-1 Fab fragment bound by the PA tag peptide, and deduced precise binding
mechanism at atomic resolution. The wealth of biochemical and structural information available will make
PA tag a very powerful protein purification and detection tool in the field of protein science.
POST 12-206
Computational Design Of Protein-DNA Nanowires
Yun Mou
Caltech, Pasadena, California, US
Computation protein design (CPD) has been successfully used to create various functional proteins,
including enzymes, protein binders, ligand binders, and protein self-assemblies. The ability to rationally
design molecular self-assembly using biological macromolecules is of particular interest because of the
potential for applications in biotechnology and medicine. Sophisticated single-component nanostructures
composed exclusively of nucleic acids or proteins have been demonstrated, but despite these successes,
the development of hybrid self-assemblies of nucleic acids and proteins via non-covalent interactions
remains
elusive.
Here, we used CPD to create a protein-DNA complex that can self-assemble into nanowires. To achieve
this, a homodimerization interface was engineered onto the Drosophila Engrailed homeodomain (ENH)
transcription factor so that the complex could bind two DNA molecules. The homodimerization interface
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POSTER ABSTRACTS
was designed de novo, whereas ENH’s native DNA-binding interface was exploited to bind a specific
double-stranded DNA (dsDNA) motif. When dsDNA fragments containing two protein-binding motifs on
opposite faces of the DNA were combined with the engineered ENH homodimer, the two components
self-assembled to form protein-DNA nanowires. Atomic force microscopy showed that the diameter of
the nanowire is approximately 10 nm, which is consistent with the length of the dsDNA fragment. The
length of the nanowire is up to 300 nm. A protein-DNA co-crystal structure confirmed that the nanowire is
formed via the designed interactions.
POST 12-207
Reversible Modification Of The N-Terminal Cysteine Residue Of Proteins Using Pyruvic Acid
Analogs
Pradeep Budhathoki, Youngha Ryu
Chemistry, Texas Christian University, Fort Worth, Texas, US
The N-terminal cysteine residue of proteins can selectively react with pyruvate analogs at pH 7 to form
their corresponding 2-methyl-2,4-thiazolidinedicarboxylic acid derivatives. Aminoxy-containing reagents
such as methoxylamine can easily reverse this reaction to regenerate the free cysteine residue. Novel
pyruvate analogs were prepared with affinity (biotin) and fluorescent (pyrene) labels, which should be
useful for the protein purification and detection, respectively. Also synthesized were pyruvate analogs with
unique chemical handles such as azide and propargyl groups. These functional groups can be used to
further modify proteins using bioorthogonal reactions without interfering with native biochemical
reactions. All of the prepared pyruvate analogs were successfully tested for the selective and efficient Nterminal modification of the Z-domain as a model protein. The modification can be easily removed to
regenerate the intact unmodified proteins.
POST 12-208
Abdesign: Computational Antibody Design Switching Species Preference And Humanizing An
Inhibitory Antibody
Assaf Alon, Sarel J. Fleishman
Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel
Monoclonal antibodies are the realization of Paul Ehrlich’s dream of a magic bullet, a drug that binds its
cognate target with high specificity. The most established method for generating monoclonal antibodies,
hybridoma technology, although very successful, suffers from limitations. Such limitations include the
difficulty in targeting a specific binding site on the desired antigen, and the inability to generate
antibodies against self antigen. Another common method for generating monoclonal antibodies is
screening libraries of antibody fragments. This method is limited by library size and quality. Furthermore,
libraries are typically restricted in their repertoires to constant complementarity-determining region (CDR)
lengths and thus sample only a fraction of conformational space. Computational design of antibodies is a
third alternative that can potentially alleviate some of these difficulties. In our laboratory we use the
rosetta modeling software suite to generate new antibodies by grafting CDRs from a database of highresolution antibody structures onto a desired framework. I use these computer-generated antibodies to
switch the species specificity of an inhibitory murine monoclonal antibody targeting the human secreted
disulfide catalyst Quiescin Sulfhydryl Oxidase (QSOX). Due to self-tolerance this antibody is nonreactive
towards the murine version of QSOX although the epitope is highly conserved. In a related project I aim
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POSTER ABSTRACTS
to humanize the inhibitory antibody. QSOX has been implicated in various types of metastatic cancer, and
a humanized version of the inhibitory antibody is of putative therapeutic value. I use rosetta to thread all
same-length human germline genes onto the murine antibody backbone. I then assess which sequence
will fold into the correct structure and place the crucial interacting residues in the same orientation as the
original antibody.
POST 12-209
Sequence-Specific Cleavage Of Initiating Methionine And Rimj-Catalyzed N-terminal Acetylation Of
The Z-domain In escherichia Coli
Youngha Ryu1, Lina Bernal-Perez2
1
Department of Chemistry, Texas Christian University, Fort Worth, Texas, US, 2Department of Chemistry
and Biochemistry, Texas Wesleyan University, Fort Worth, Texas, US
The present study used the Z-domain as a model protein to establish the sequence dependence of the
Met1 cleavage and RimJ-mediated N-terminal acetylation in E. coli. The Z-domain variants differing by the
second or third amino acid residue were expressed and analyzed by mass spectrometry. The Met1 residue
of the Z-domain was removed only when the second amino acid was glycine, alanine, proline, serine,
threonine, cysteine, or valine, consistent with the reported sequence specificity of the E. coli MAP. Only
subsequent to the Met1 cleavage, the RimJ-catalyzed N-terminal acetylation mainly occurred at the Nterminal serine, threonine, or valine residues. The N-terminal acetylation of the Z-domain was significantly
decreased by glycine, proline, arginine or lysine in the penultimate position, but was enhanced by
hydrophobic or negatively charged residues in the same position. Practically, this study offers a basis to
predict or control Met1 cleavage and N-terminal acetylation of recombinant proteins in E. coli, especially
when these N-terminal end modifications significantly affect protein stability or activity.
POST 12-210
In Vitro Selection And Evolution Of Membrane Proteins Using Liposome Display
Tomoaki Matsuura, Satoshi Fujii, Yasuaki Kazuta, Takeshi Sunami, Tetsuya Yomo
Osaka University, Suita, Osaka, Japan
An in vitro translation (IVT) system produces proteins without using living cells. Based on this
characteristic, IVT has been used for various applications including in vitro protein evolution. As protein
synthesis with IVT is disconnected from cell growth, a wide range of proteins can be targeted. However,
the proteins evolved to date using in vitro evolutionary methods have all been globular proteins (e.g.,
antibodies and enzymes), and there have been no previous reports of in vitro evolution of membrane
proteins. Membrane proteins account for 20–25% of all open reading frames in the genome, and more
importantly, more than 50% of the current pharmaceutical targets are membrane proteins. Despite their
importance, technical difficulties such as the expression of membrane proteins in heterologous host cell,
and establishing a functional screening assay for each membrane proteins, have limited the usage of
directed evolution for engineering membrane proteins. Here, we report the development of a method,
named liposome display, for evolving the properties of membrane proteins entirely in vitro which will
eliminate these difficulties and thus enable the more rapid and efficient evolution of a greater variety of
membrane proteins. This method, which involves in vitro protein synthesis inside liposomes, was applied
to evolve the pore-forming activity of alpha-hemolysin (AH), a membrane protein derived
from Staphylococcus aureus. The obtained AH mutant possessed only two point mutations but exhibited
a 30-fold increase in its pore-forming activity compared with the wild-type. Given its high degree of
controllability, the ability to modify protein synthesis, and functional screening conditions, liposome
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POSTER ABSTRACTS
display makes possible the rapid and efficient evolution of a wide range of membrane proteins, including
transporters and signaling proteins, thereby boosting the field of membrane protein engineering.
POST 12-211
Ubiquibodies: Engineered E3 Ubiquitin Ligases for Targeted Degradation
Erin A. Stephens1, Alyse D. Portnoff2, Morgan R. Baltz2, Jeffrey D. Varner2, Matthew P. DeLisa2
1
Biochemistry, Molecular & Cell Biology, Cornell University, Ithaca, New York, US, 2Biochemical &
Chemical Engineering, Cornell University, Ithaca, New York, US
Protein silencing is a powerful tool for both studying the function of proteins in cells and eliminating
disease-causing peptides from a system. Current methods for protein silencing act at the DNA and RNA
levels; however, these methods cannot be used to interrogate the difference between isoforms of a
single protein. Pools of protein isoforms are most commonly generated by post-translational
modifications, such as phosphorylation, cleavage, ubiquitination, etc. In order to study proteins at a
post-translational level, we are developing a silencing technology to function at the protein level. Cells
naturally turn over proteins via the ubiquitin-proteasome pathway (UPP); by hijacking this pathway,
non-native substrates can be targeted to the UPP for degradation, thus silencing protein function. The
canonical UPP pathway is composed of three enzymes: E1 (ubiquitin activating enzyme), E2 (ubiquitin
conjugating enzyme), and E3 (ubiquitin ligating enzyme). The E3 ubiquitin ligase determines the target
specificity and is the focus of our design. Here we present a family of rationally-designed E3s, termed
ubiquibodies, composed of an endogenous ubiquitin ligase catalytic domain fused to variable synthetic
binding domains. The catalytic domain is the U-box domain of C-terminus of Hsc70-interacting protein
(CHIP). In order to endow the ubiquibodies with tunable specificity, CHIP’s natural binding domain was
replaced with synthetic binding domains, including a single chain variable fragment (scFv), a fibronectin
type three domain (FN3), and designed ankyrin repeat proteins (DARPins). We first show that
ubiquibodies can ubiquitinate novel substrates in vitro, and that the same substrates can be silenced in
mammalian cells expressing both the target and ubiquibody. We then show the ubiquibodies can be
similarly engineered against endogenous proteins. Ongoing work aims to generate ubiquibodies that can
differentiate between phosphorylated and non-phosphorylated isoforms of the same protein,
specifically the map kinase ERK2. And while the current system successfully silences targets, we
hypothesize that delineating the structural and kinetic properties of ubiquibodies will enable calculable
functionality. We conclude that ubiquibodies can be used as a customizable platform to silence proteins
at the post-translational level in mammalian cells.
POST 12-212
Engineering Ubiquitin To Recognize Non-Cognate Proteins Implicated In Cancer
Isabel Leung1, 2, Sachdev Sidhu1, Nick Jarvik1
1
Donnelly Centre of Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario,
Canada, 2Department of Molecular Genetics , University of Toronto, Toronto, Ontario, Canada
Understanding and manipulating protein-protein interactions is essential to fully comprehend basic cell
function and disease states. However, a complete set of affinity tools for probing protein function is
sorely lacking. We have employed an ambitious design strategy to produce a high quality Ubiquitin (Ub)
phage display library for isolating affinity reagents to a wide array of protein folds that do not normally
2
bind Ub. We identified the native interaction surface on Ub, a relatively flat 2000Å beta sheet, as a good
candidate for engineering novel protein interactions. First, we employed a phage displayed based
mutation tolerance scan to identify surface exposed residues on the beta-sheet that are structurally
sensitive to mutation, and thus were kept as WT in the library design. Subsequently, using a minimalistic
randomization scheme previously utilized in antibody loop engineering, we randomized 24 of the 76 Ub
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POSTER ABSTRACTS
residues with 6 amino acids that have functioned well in synthetic antibody libraries. In addition, to
increase the stability of the library variants, stability selection was integrated into library construction.
Using the resulting library, we were able to generate binding Ub variants (UbV) to 17 of 29 proteins tested
on the first trial. Validation of UbVs binding to two distinct targets, Grb2 SH2 domain and Her3
extracellular domain, are currently underway. Grb2 is an adaptor protein that relays RTK stimulation to
transcriptional programs, and Her3 is a member of the EGFR family where overexpression has been
implicated in cancers. UbVs binding to Grb2 or Her3 show specificity to the intended target and do not
recognize closely related family members by ELISA. We have intracellularly expressed Grb2-binding UbVs
in mammalian cells and observed physical interaction to Grb2 by co-immunoprecipitation. Our SPR data
reveal GST tagged Grb2 binding- UbVs have single digit nM affinity to Grb2, which is comparable to
antibody-antigen interactions. As for Her3 binding UbVs characterization, we have observed antagonistic
Her3 receptor signaling effects on SKBR3 breast cancer cells lines that are known to overexpressed Her3.
In all, we have engineered UbVs that can behave like antibody affinity reagents to influence membrane
receptor signaling and have the potential to be used as intracellular affinity tools for perturbing proteinprotein interactions when expressed inside mammalian cells.
POST 12-213
Optimization Of A Protein Labelling Technique For Fluorogenic, X-Ray Crystallography And NMR
Applications
Miroslava Strmiskova, Natalie K. Goto, Jeffrey W. Keillor
Chemistry, University of Ottawa, Ottawa, Ontario, Canada
Fluorescent protein labelling is a powerful tool for the sensitive visualization of proteins in living cells,
allowing the elucidation of their localization, trafficking and ultimately their cellular function. We have
developed a novel labelling technique based on the genetic fusion of a protein of interest to a small
helical peptide sequence containing two Cys residues (dC10). This tag can undergo an efficient reaction
with small fluorogenic labelling agents composed of a fluorophore and a dimaleimide core (dM10) that
confers high reaction specificity, and quenches the latent fluorescence through photo-induced electron
transfer, until both of its maleimide groups have form robust covalent bonds with the tag Cys thiol
groups. Our initial efforts at intracellular protein labelling demonstrated the importance of the selectivity
of the labelling reaction, which is dependent on the reactivity of the dC10 tag. To that end, we reengineered the dC10 tag through semi-rational protein design. Mutant libraries were prepared through
combinatorial mutation at specific positions of the helical tag sequence, and screened for their
fluorogenic reactivity. In this way, we identified a novel sequence for a next-generation dC10 tag that
confers 10-fold greater selectivity. Subsequent mechanistic studies revealed the basis for this dramatic
increase in reactivity. Current applications of this powerful labelling technique will also be discussed. In
addition to the fluorescent labelling of specific proteins in living cells, these include the site-specific
chelation of lanthanide ions for NMR spectroscopy and site-specific covalent heavy-atom labelling for Xray crystallography.
POST 12-214
Computational Design of Cystatin and Ketosteroid Isomerase Folds from Scratch: From Structure
to Self-Labeling Function
Enrique Marcos, Dr. David Baker
Biochemistry, University of Washington, Seattle, Washington, US
1
The principles and computational methods for designing ideal protein structures have now made
possible the de novo design of small and thermostable proteins with different folds. This major
breakthrough now enables control over the protein backbone to customize protein structures for hosting
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POSTER ABSTRACTS
a variety of catalytic active sites. In this contribution, we have computationally designed and
experimentally tested de novo proteins with the cystatin and Ketosteroid Isomerase (KSI) fold. A curved βsheet builds a large and accessible cavity in the KI fold that makes it very attractive for the design of small
2
molecule binders and enzymes . As a proof of concept, we first designed from scratch the minimalist
version of a curved β-sheet in the cystatin fold. Subsequently, we have successfully designed thermostable
and monomeric proteins in the KSI fold. As a first application of these de novo KI folds, we have designed
active sites with a nucleophilic lysine for the formation of an enamine with a fluorescent probe. This selflabeling function should be useful for image monitoring of proteins in live cells. 1. Koga N. et al.,
Nature, 2012, 491, 222 2. Tinberg CE. et al., Nature, 2013, 501, 212
POST 12-215
Development of Novel p16INK4a Peptide Mimetics as Anticancer Therapy
Marian Kratzke2, Yuk Sham3, Mark A. Klein1
1
Medicine, Minneapolis VA Healthcare System and University of Minnesota, 2Research Service,
Minneapolis VA Healthcare System, 3Center for Drug Design, University of Minnesota, Minneapolis,
Minnesota, US
Introduction: Mesothelioma is a highly fatal disease that has poorly effective therapy with dose-limiting
side-effects. Low expression of the endogenous CDK4/CDK6 inhibitor, p16INK4a, has been demonstrated
in up to 90% of mesothelioma tumors. Replacement of p16INK4a activity via gene therapy in laboratory
models has demonstrated activity against CDK4 and CDK6, tumor response, and an increase in survival in
xenograft models. Two truncated peptides (FLDTLVVLHR and DAAREGFLDTLVVLHRAGAR) derived from
rd
the 3 anykyrin repeat of p16INK4a have been shown to exhibit similar activity to the full-length
protein. An isolated 10mer peptide has also been shown to maintain its native helical structure away from
its full length protein. Hypothesis: Protein-protein interactions between CDK4/6 and p16INK4a can be
replicated or disrupted by engineered stabilized helical peptides identified from shortened peptides that
interact with CDK4/6. Results and Discussion: We evaluated the bioactivities of several truncated
peptides against CDK4/6 and mesothelioma cell lines. The IC50 values against CDK4 and CDK6 for the
native peptides ranged from 14.6 μM to 21.2 μM. Peptides with a TAT-leader sequence (YGRKKRRQRRR)
bound to the amino-terminal end exhibit a wider range of IC50 activities from 860 nM to 257
μM. Stabilized helical peptide derivatives (one olefin linker per peptide, i,i+4) were also studied, and the
IC50 values ranged from 49 -105 μM. Against mesothelioma cell lines, the IC50 values against TAT-derived
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POSTER ABSTRACTS
peptides ranged from 37.6 to 88.2 μM, while IC50 values for all the other peptides were > 1mM. We are
currently engaged in molecular dynamics simulations to determine whether a correlation persists between
the percent helicity of these peptides and its bioactivity. We anticipate such a correlation will greatly
enrich our understanding in the transient structural nature of our peptides and will provide an improved
platform for further design of stabilized helical peptide candidates.
POST 12-216
A New Protein Cage Architecture Formed Via Gold Cluster Catalysis
Ali D. Malay1, Kenji Iwasaki2, Zuben Brown2, Jonathan G. Heddle1
1
RIKEN, Wako, Saitama, Japan, 2Department of Biological Sciences, Osaka University, Osaka, Osaka,
Japan
Protein cage structures hold great interest in the field of bioengineering as a platform for the
development of advanced functional materials that include drug delivery devices and nano reaction
vessels. We have produced a new type of protein cage structure based on the ring-shaped undecameric
TRAP protein as building block, which we have termed TRAP-CLS. Several distinct features of TRAP-CLS
will be discussed. The hollow, spherical protein shells are held together by disulfide crosslinks, with cage
assembly proceeding via thiol oxidation using gold nanoparticle catalysis. Significantly, despite the
stability of TRAP-CLS, it can readily be broken up in response to changes in redox conditions. Structural
analyses have revealed that TRAP-CLS is composed of 24 TRAP rings and exhibits cuboctahedral
symmetry. Intriguingly, the ring units are arranged in a chiral snub-cube configuration, the first reported
case in a protein cage structure.
POST 12-217
Identification Of Residues In TIMP-1 That Are Critical In Interaction Between MMP-9 And TIMP-1.
Ruiying Wang, Alexandra Hockla, Evette S. Radisky
Mayo Clinic, Jacksonville, Florida, US
Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family of zinc-dependent endopeptidases
that are responsible for degradation of the extracellular matrix (ECM) in normal and pathological
conditions. MMP-9 has been linked to many pathological processes including cancer, cardiovascular,
neurologic and inflammatory diseases, and is an attractive target for therapy development. Tissue
inhibitor of metalloproteinases-1 (TIMP1) is a natural and potent protein inhibitor of MMP-9. TIMP-1
possesses the unique ability to bind not only to the catalytic domain but also to the hemopexin-like
domain of MMP-9 (PEX9); this high-affinity interaction contributes to the selectivity of TIMP-1 toward
MMP-9. The epitopes involved in this interaction have not yet been identified, which represents a barrier
to our understanding of how TIMP-1 regulates MMP-9. Here, we report studies to map the protein
epitopes involved in the interaction of TIMP-1 with PEX9. Because TIMP-2, a paralogue of TIMP-1, does
not form an analogous interaction with PEX9, we reason that the protein residues responsible for these
interactions should be conserved among TIMP-1 orthologues, but show dissimilarity with the
corresponding residues in TIMP-2 proteins. From a multiple sequence alignment of mammalian TIMPS,
we identified residues of TIMP-1 that meet this criterion, and we made a series of TIMP-1 mutant proteins
in which these candidate residues were mutated to the corresponding residues in TIMP-2. To test the
effect of each mutation on the binding affinity of TIMP-1 toward PEX9, we used the Octet Red 384
platform for biolayer interferometry-based measurement of protein-protein association and
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POSTER ABSTRACTS
dissociation. Our results obtained from the Octet Red 384 platform confirm prior reports that the Cterminal domain (CTD) of TIMP-1 is responsible for the high affinity interaction with PEX9, since a TIMP-1
chimeric protein possessing the TIMP-2-CTD shows very low-affinity binding to PEX9, comparable to that
observed for TIMP-2. We further find that multiple discontiguous sequence and structural elements
within the TIMP-1 CTD are important in the binding interaction with PEX9, and we identify a number of
key residues that appear to play particularly critical roles in the binding. These insights into TIMP-1
structure and function may be valuable for future efforts to engineer therapeutic MMP-9-selective
inhibitors based on the TIMP-1 scaffold.
POST 12-218
Computationally Designed Green Fluorescent Protein Based Biosensors
Shounak Banerjee1, 4, Yao-Ming Huang2, Donna E. Crone1, Diana I. Paredes3, Jonathan S. Dordick3, 1, 4,
Christopher Bystroff1, 5, 4
1
Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 2Bioengineering and
Therapeutic Sciences, University of California at San Francisco, San Francisco, California, US, 3Chemical
and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, US, 4Center for
Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York,
US, 5Computer Science, Rensselaer Polytechnic Institute, Troy, New York, US
A leave-one-out GFP (LOO-GFP) is a circularly permuted (cp) green fluorescent protein that has a strand
omitted to create a pocket. The empty pocket may be re-shaped by computational protein design to bind
a foreign peptide and reconstitute GFP's fluorescent function. In this work, LOO7-GFP, with strand 7 left
out, was computationally designed to bind a peptide (HA) representing a specific antigenic determinant
on H5N1 influenza virus haemagglutinin, using in-house software called DEEdesign. The resulting library
of variants was co-expressed with the target peptide HA and colonies were selected for
fluorescence. Selected variants were purified for biochemical analysis. One of these, LOO7-GFP: HA4
fluoresced in the unbound state, as did wild-type LOO7-GFP. But LOO7-GFP: HA4 unexpectedly lost
fluorescence upon addition of synthetic HA peptide, whereas wild-type LOO7-GFP doubled in
fluorescence when the wild-type strand 7 peptide was added. Refolding of His-tagged LOO7-GFP:HA4
and wild-type LOO7-GFP on Ni-NTA beads eliminated most of the auto-fluorescence, suggesting that
dimerization was involved in autofluorescence, and that peptide binding blocked dimerization. But
binding to the computational variant was too weak to form the monomeric reconstituted state, leading to
loss of fluorescence. To better understand LOO-GFPs in the unbound state, we have evaluated circular
dichroism spectra for LOO-GFPs 4, 7, 8, 9 and 11. Although data suggest that LOO8-GFP and LOO11-GFP
have the most native-like structure in the bound state, computational design attempts on the LOO11-GFP
scaffold again produced highly destabilized and non-fluorescent proteins. No fluorescent colonies were
found in the same library, even if the target peptide (a sequence from dengue virus NS1 protein) was
covalently attached (called "leave-it-in" GFP, or LII-GFP). Analysis of the non-fluorescent designed
sequence libraries in the context of the chromophore maturation pathway is helping us to understand the
sequence requirements for the formation of the GFP chromophore.
POST 12-219
Atomic Force Microscopy Characterization of Beta-Solenoid Based Amyloid Fibrils
Arpad Karsai2, Maria D.R. Peralta2, Alice Ngo2, N. Robert Hayre3, 1, Nima Mirzaee3, 1, Alexander J. Kluber1,
Xi Chen2, Gang-yu Liu2, Michael Toney2, Rajiv R.P. Singh3, 1, Daniel L. Cox3, 1
1
Institute for Complex Adaptive Matter, University of California, Davis, California, US, 2Department of
Chemistry, University of California, Davis, California, US, 3Department of Physics, University of California,
Davis, California, US
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POSTER ABSTRACTS
Spruce budworm sbwTHP and perennial ryegrass, Lolium perenne LpAFP antifreeze proteins have left
handed beta solenoid structures and are inhibited from in vivo aggregation by a capping structure or
distortion. Using rational design, these proteins have been modified in order to facilitate their selfassembly to amyloid fibrils. To create the mutant sbwTHP, termed NoDS9, all cysteines were removed and
replaced with serine residues, the capping motif at the C-terminus was deleted and two monomers were
genetically fused together. Salt bridges were added to the interfaces to aid in polymerization. A similar
rational was used to design for the engineered amyloid formation of mutant LpAFP, termed
3ULT_m1. Structurally, wild type LpAFP has short distorted ends composed of 5 amino acids in the Nterminus and 9 amino acids in the C-terminus, in addition to a slight bulge in three of the rungs near the
N-terminus which were both deleted in the design for 3ULT_m1. The designed LpAFP were left as
monomers however salt bridges were added to promote amyloid fibril formation.
Atomic force
microscopy (AFM) characterization of NoDS9 and 3ULT_m1 revealed that both mutants can form
unbranched fibrils. The fibrils are Thioflavin-T positive. NoDS9 first form 2.5 nm tall, micrometer long
protofilaments within 48 hours. Afterwards, the protofilaments start lateral association and further
elongation forming regular amyloid fibrils with 5.0 to 15.0 nm height showing that NoDS9 follows the
classic amyloid formation pathway. Two very distinct type of fibrils of 3ULT_m1 were revealed by AFM.
Type 1 fibrils are 1.0 nm tall with low (0.35μm) persistence length and type 2 fibrils with 1.6 nm height and
higher (0.7μm) persistence length. Our results indicate that rational design and modification of the tertiary
structure of wild type sbwTHP and LpAFP beta solenoid proteins results in amyloid fibrils proving that our
method can be a platform to create new self-assembled biomaterials. Our long term goal is to create
such controlled self-assembly systems which can be used for nanoparticle templating and synthesis.
POST 12-220
Reverse Protein Engineering Towards a Fluorescent Peptide
Zhiwen J. Zhang, Blake Williams
Bioengineering, Santa Clara University, Santa Clara, California, US
The green fluorescent protein (GFP) is a powerful tool used as a fused protein reporter in biological and
biomedical studies. However, sometimes its applications, especially in vivo diagnostic studies, are limited
due to its large molecular size. Herein, we report a “Reverse Protein Engineering” methodology to evolve
a peptide with function retained from its parent protein. Using the full length GFP as a template, a 74
amino acid peptide (iGFP) has been discovered that emits fluorescent signals. iGFP shares the same
chromophore sequence as GFP, but interestingly displays different excitation and fluorescent emission
spectra from the full length GFP. The methodology of “Reverse Protein Engineering” could be a general
protocol used to develop peptides with functions such as catalytic peptides and therapeutic peptides
etc. – a peptide world.
POST 12-221
Beyond Glutaraldehyde: The Search for Optimal Chemical Crosslinkers for Protein Crystal
Thaddaus R. Huber, Jacob Sebesta, Christopher Snow
Colorado State University, Fort Collins, Colorado, US
Protein crystals are porous three-dimensional self-assembling materials with angstrom level precision.
Currently, there is interest in using protein crystals as a platform for industrial catalysis, drug delivery, and
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POSTER ABSTRACTS
scaffolds to template inorganic nanomaterials. Due to the fragility of protein crystals, a common method
for increasing stability is chemical crosslinking via glutaraldehyde. However, glutaraldehyde is notoriously
reactive and can disrupt crystal quality. Here we quantify the performance of a panel of alternative
crosslinkers with aldehyde, N-Hydroxysuccinimide (NHS), and imidoester active groups to stabilize hen
egg-white lysozyme (HEWL) crystals. Stability and x-ray diffraction resolution of crystals treated with the
various crosslinkers were compared. Computational identification of potential crosslinks using X-LINKR, a
custom protein crystal crosslinking predictor, was compared to experimental results to determine if
crosslinker performance could be predicted a priori. The work herein lays the groundwork for rational
design of protein crystal modifications that improve stability via chemical fixation while retaining crystal
quality.
POST 12-222
Utilization of Collagen IV NC1 Domains to Control Helical Composition: A Recombinant Strategy
for the Production of Collagen IV Protomers
Kyle L. Brown, Vadim Pedchenko, Selene Colon, Tim Blackwell, Ambra Pozzi, Billy Hudson
Vanderbilt University, Nashville, Tennessee, US
The interaction of basement membrane collagen IV and integrin cell-surface receptors is a fundamental
element of the reciprocal dialogue that governs the structure and function of normal tissues.
Unfortunately, the analyses of collagen IV network formation and extracellular integrin interactions
have been hampered by a lack of collagen reagents of definite composition. Tissue sources for collagen
IV are plagued with issues of content, solubility, yield, and purity. Collagen "tool-kits", comprised of
synthetic peptides designed to self-assemble into homotrimers, have facilitated significant advances in
the analysis of collagen II and III interactions. However, this strategy is not amenable to the study of
heterotrimic collagen IV. As a novel approach to overcome the limitations of tissue sources, a
recombinant collagen IV protomer has been generated in mammalian HEK293 cells. Collagen chain
composition and register were mediated by the incorporation of C-terminal collagen IV NC1 domains
within the recombinant constructs. Western-blot, size-exclusion chromatography, multi-angle light
scattering, and ELISA determined the molecular composition of the recombinant collagen IV protomer
to be comprised of two α1 and one α2 collagen IV chains (residues 1121-1730) with an apparent
molecular mass of 239.4 kD. Circular dichroism indicated the collagenous domain remained helical up to
30°C while the NC1 domains were thermostable to 66°C. The GFOGER integrin-binding motif was sitespecifically engineered into the collagenous domain (residues 1460-1472). Proper collagen chain register
was confirmed by the functional ability of the recombinant protomer to bind integrin α2β1 as
determined by solid-phase binding to recombinant integrin I-domains and HT1080 cellular adhesion
assays. Functional blocking antibodies confirmed that HT1080 cell adhesion to recombinant collagen IV
protomer was predominantly mediated by integrin α2β1. This report demonstrates the ability to
generate collagen IV biomolecules of known composition in a scalable, cost effective manner for the
study of collagen IV network assembly and extracellular receptor interactions.
POST 12-223
Secondary Antibody Cross-Reactivity Identification Using ProteOn XPR36 System
Gary Ross, Perry Ripa, Mohammed Yousef
Bio-Rad Laboratories, Hercules, California, US
nd
Species-specific secondary antibodies (2 Abs) are essential for reliable immunological assays, utilized in
nd
the biological sciences. We utilize the ProteOnXPR36 to rapidly characterize the binding of 2 Ab samples
to various species IgGs. Goat, human and mouse IgG samples are coupled to parallel channels on a
161
POSTER ABSTRACTS
ProteOn GLC chip. After rotation of the ProteOn multi-channel module to the horizontal orientation, goat
nd
anti-mouse-Fc (GAM) and goat anti-human-Fc (GAH) 2 Abs (six varying concentrations) are then injected
across the primary Ab surfaces, providing kinetic characterization of species-specific binding and
comparative quantitation of non-specific (cross-reactive) binding. GAM (3 ng/ml – 80 ug/ml) binds only
to mouse IgG. In contrast, GAH (> 60 ng/ml) shows cross-reactivity with mouse IgG. GAH profiles show
slower association (ka) and faster dissociation (kd) rates for binding to mouse IgG, compared to the
specific human IgG surface. Overlay of comparative sensograms provides an estimate of 0.4% (GAH to
mouse IgG) cross-reactivity. Longer association times (10-16 min) can detect cross-species binding in the
nd
0.1% - 0.5% range. ProteOn Manager Concentration Analysis uses initial binding slopes of 2 Ab
nd
standards to estimate cross-reactive 2 Ab concentrations in unknown samples. Quantitation (at < 5%
nd
error) for 2 Abs below 50 ng/ml is demonstrated.
Poster Session: Cellular Structures
POST 02-224
Dissecting the repetitive C-terminal domain of RNA polymerase II
Stephen Fuchs, Mohammad Mosaheb, Summer M. Morrill
Biology, Tufts University, Medford, Massachusetts, US
Tandemly repeating amino acid sequences play diverse, functionally important roles in the cell. One in
particular, the C-terminal domain (CTD) of RNA polymerase II (RNAPII) plays an essential role in
coordinating processes such as mRNA processing with ongoing transcription. Protein factors recognize
specific phosphorylated forms of a tandemly-repeating seven amino acid consensus sequence
(YSPTSPS). This sequence is conserved across most eukaryotes and varies in length from 26 repeats
(yeast) to 52 repeats (humans). This work attempts to explore a very long-standing question regarding
CTD function – do repeats have overlapping function or do they show some level of specificity based on
either sequence or proximity to the rest of the RNA polymerase II complex. Here we present evidence that
repeats indeed show independent function.
POST 02-225
Bacterial Microcompartments: Substrate Transport through Shell Pores
Sunny Chun2, 1, Chiranjit Chowdhury3, Jiyong Park1, Kendall N. Houk1, Thomas A. Bobik3, Todd O. Yeates2,
1
1
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles,
California, US, 2Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California,
US, 3Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, US
Bacterial Microcompartments (BMCs) are proteinaceous organelles that sequester key metabolic reactions
to increase enzymatic efficiency and prevent the loss of volatile or toxic intermediates. Operons encoding
at least seven apparently metabolically distinct BMCs have been identified in 20% of bacteria, including
BMCs involved in utilizing inorganic carbon (i.e. bicarbonate), 1,2-propanediol, and ethanolamine.
Ranging from 100 to 150 nm in diameter, BMCs are encapsulated in a protein shell consisting of
hexagonal tiles of the conserved BMC-fold proteins. The BMC-fold proteins homo-oligomerize into
hexagonal or pseudo-hexagonal assemblies with unique functions; most possess central pores presumed
to be involved in transport of substrates and products. The mechanisms of small molecule transport
through, and retention of toxic intermediates by, BMCs remain poorly understood. We hypothesize that
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POSTER ABSTRACTS
the BMC shell pores serve a role for molecular transport by passive selective diffusion. One such poreforming BMC-fold protein is PduA of the Propanediol utilization (Pdu) BMC. Here, we present the high
resolution crystal structures of PduA pore mutants S40Q and S40L, which support their observed
phenotypes of increased and decreased BMC activity, respectively (unpublished). Initial findings using
Molecular Dynamics to investigate the free energy of small molecule penetrance through the PduA pore
(r=2.8Å) also indicates a higher free energy barrier for the toxic intermediate propionaldehyde than for
substrate 1,2-propanediol (unpublished). Using both biochemical and computational approaches, we
identify the importance of the key residue S40 and hydrogen-bonding of the small molecule metabolites.
Our experiments shed insight on BMC shell pore dynamics and function, furthering our understanding of
BMC systems.
POST 02-226
Sequence Signature for Recognition of Histone H3 Arginine (R2)
Francisca Essel, Suvobrata Chakravarty, Tao Lin
Chemistry and Biochemistry, South Dakota State University, Brookings, South Dakota, US
Recognition of covalently modified as well as unmodified N-terminal histone residues by specific factors
play a crucial role in regulating chromatin structure and assembly. Hence, a large effort in the past
decade has gone into discovering protein factors that read (recognize) site-specific histone peptide
segments. However, majority of these have focused on the recognition of covalently modified histone
amino acids (e.g. methylated, and acetylated Lysine) while there are fewer reports on the recognition of
unmodified histone Lysine and Arginine. Here we focus on identifying protein factors capable of
recognizing unmodified histone H3R2. Sequences of human PHD (plant homeodomain) family having a
distinct pattern of negatively charged residues that are predicted to form salt-bridges with H3R2 are
tested for site-specific binding using a fluorescent protein based colorimetric assay. We observed that
the first PHD finger domain of Lysine-specific Demethylase 5B (KDM5B-PHD1) strongly interacts with
H3R2 with 8.85 mM while H3K4 contributes negligibly to the interaction of unmodified histone H3-1-11
residues i.e. KDM5B-PHD1 belongs to a PHD family subgroup that is distinct from the subfamily specific
for the recognition of unmodified H3K4. Consistency of the binding result with that of ITC suggests a
simple assay to probe protein-protein interaction.
POST 02-227
Microfluidic Control And In Situ Monitoring Of Microtubule Maturation Kinetics Provide Evidence
For A New Stabilising Cap Model Of Dynamic Instability
Christian Duellberg1, Nicholas Cade1, David Holmes2, Thomas Surrey1
1
Microtubule Cytoskeleton Lab, Cancer Research UK, London Research Institute, London, United
Kingdom, 2London Centre for Nanotechnology, University College London, London, Greater London,
United Kingdom
Switching between growth and shrinkage is an important property of microtubule ends. The molecular
mechanism underlying this switch is still not well understood. Using a novel microfluidics-assisted total
internal reflection fluorescence microscopy assay, we directly tested predictions of the most prominent
current models of dynamic instability. We manipulated in a controlled manner GTPase cycle-associated
transition kinetics and directly linked these kinetics to microtubule stability. With these new tools in hand
and in combination with rapid tubulin concentration changes, we found that the mechanism of
catastrophe induction cannot be explained satisfactorily by recently proposed models. We further
demonstrate that the density and the turnover (but not the length) of a conformational cap recognized by
EB1 family proteins determines filament stability and that fluctuations of the cap density are directly
linked to momentary filament stability. Our model for catastrophe induction effectively combines
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POSTER ABSTRACTS
properties of previous coupled and stochastic GTP cap models and takes structural changes at
microtubule ends into account. It explains microtubule lifetime distributions under steady state and after
rapid tubulin wash-out. Our results provide a unified conceptual framework that explains the molecular
mechanism of catastrophe induction.
POST 02-228
Investigating The Function Of Suppressor Of IKK-epsilon
Sean W. McKinley2, Kenneth F. Lawrence2, Jessica K. Bell1
1
Biochemistry, Virginia Commonwealth University, Richmond, Virginia, US, 2Microbiology &
Immunology, Virginia Commonwealth University, Richmond, Virginia, US
Pathogen challenge elicits an immediate response by the innate immune system. Several innate immune
signaling pathways converge to activate TANK binding kinase 1 (TBK1), a ubiquitously expressed kinase.
TBK1 phosphorylates substrates that mediate pro-inflammatory cytokine and type I interferon production
or sequestering of intracellular bacteria. We have defined a new TBK1 substrate, Suppressor of IKKepsilon
(SIKE). With respect to other TBK1 substrates, SIKE acted as a nanomolar, mixed-type inhibitor of TBK1.
Although SIKE could influence TBK1 activity as a high affinity substrate, the primary function of SIKE
remains unknown. Preliminary co-immunoprecipitation assays of SIKE suggested that the SIKE interaction
network impinged upon the cytoskeleton and RNA transport. We hypothesize that SIKE links innate
immune signaling to mRNA transport. To further characterize SIKE’s function within the antiviral response,
we used immunofluorescence assays to identify colocalization between SIKE and cellular markers in
epithelial and myeloid cell types. Colocalization was quantified at the level of whole cell or region of
interest. SIKE colocalized with cytoskeleton components (actin and tubulin), endosomal and plasma
membrane markers (Rab11, LAMP-1, and LC3), and ribosomes (S6). In parallel experiments to identify SIKE
function, recombinant SIKE protein purification strategies were developed to produce material for
structural analysis. Purification schemes utilizing a 6XHis tagged construct under denaturing and native
conditions with Ni-NTA or TALON resin and incorporation of ion exchange have been tested. For each
scheme, size exclusion chromatography and SDS-PAGE/Coomassie/silver stain were used to assess purity.
The optimal purification scheme targeted purification of SIKE from the insoluble pellet under denaturing
conditions using TALON resin to minimize co-purification with E. coil protein, SlyD. Funds to support these
studies provided by CCTR Endowment Fund and NIH R21AI107447.
POST 02-229
Structure Of BDBT Reveals A Role For Noncanonical FK506 Binding Protein In Regulation Of The
Fly Circadian Clock
Boadi Agyekum, Jin-Yuan Fan, Anandakrishnan Venkatesan, Jeffrey Price, Samuel Bouyain
UMKC, Kansas City, Missouri, US
In the fly circadian cycle, DOUBLETIME (DBT) is a principal kinase that phosphorylates PERIOD (PER) and
targets it for degradation. Bride of DOUBLETIME (BDBT) is a novel DBT-binding protein identified in a
proteomic screen. RNAi-mediated knock-down of BDBT produced behavioral arrhythmicity, high levels of
hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME, suggesting that BDBT is a novel
clock regulatory protein. To gain insight into the function of BDBT in the Drosophila circadian clock, we
determined the crystal structure of the first 211 amino acid residues of BDBT. Our analysis revealed that
the closest structural homologue of the BDBT N-terminus (residues 1-120) is FK506 binding protein 1A
(FKBP1A) even though they share only limited sequence identity. FKBP1A is a peptidyl-prolyl cis-trans
isomerase, but only 2 of the 13 residues involved in isomerase activity are conserved in BDBT, suggesting
that BDBT is not enzymatically active. Instead, our biochemical experiments indicate that the N-terminus
of BDBT binds to DBT. Although there is no BDBT homologue in vertebrates, sequence analyses indicate
164
POSTER ABSTRACTS
that the C-terminus of BDBT resembles the tetratricopeptide repeat region of the non-canonical FK506binding proteins FKBP51, FKBP52, and FKBPL, which bind the vertebrate DBT homologue CK1δ and CK1ε.
Thus we propose that these proteins may function as functional homologues of BDBT in vertebrates.
POST 02-230
The PKD-Related Proteins ANKS6, BICC1, And ANKS3 Form A SAM Domain Interaction Network
Catherine N. Leettola1, Mary J. Knight1, Duilio Cascio2, James U. Bowie1
1
Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California,
US, 2Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, US
Polycystic kidney disease (PKD) is the most common genetic disorder leading to end-stage renal failure in
humans. Mutations in the proteins ANKS6 and BICC1 are responsible for disease in rat and mouse models
of PKD, respectively, yet the exact mechanism of disease causation remains unknown. Both ANKS6 and
BICC1 contain SAM domains, which are known protein-protein interaction domains that are capable of
binding each other to form heterodimers, helical polymers, or sheets of polymers that can act as
scaffolds. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both
ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM
domain at the C-terminus. Using a negGFP native gel binding assay, we have identified the SAM domain
of ANKS3 as a direct binding partner of both the ANKS6 SAM domain and the BICC1 SAM domain. We
find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. Crystal
structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex reveal the
molecular details of these SAM domain associations. An R823W mutation in the SAM domain of ANKS6 is
causative of disease in the PKD/Mhm(cy/+) rat model of autosomal dominant PKD. Using circular
dichroism, we determined that this point mutation disrupts ANKS6 function by dramatically destabilizing
the SAM domain such that the interaction with ANKS3-SAM is lost. ANKS6, ANKS3, and BICC1 each
contain other functional protein domains in addition to their SAM domains. We propose that a network
of interactions between the SAM domains of ANKS3, ANKS6, and BICC1 defines a variety of scaffolds
responsible for correctly organizing a variety of protein complexes and that this network of interactions is
responsible for proper kidney development and function.
Poster Session: Chemical Biology & Enzymology
POST 03-231
The Variations Of Protein Splicing: Regulation And Mechanism Of Non-Canonical Inteins
Kenneth Mills, Julie N. Reitter, Michael Nicastri, Jennie Williams, Kathryn Colelli, Michelle Marieni
Holy Cross, Worcester, Massachusetts, US
Protein splicing is the post-translational excision of an intervening polypeptide, or intein, concomitant
with the ligation of the flanking domains, or exteins. The inteins that interrupt the PolII of Methanoculleus
marisnigri (Mma) and Pyrococcus abyssi (Pab) each facilitate protein splicing with C-terminal Gln in place
of the highly conserved Asn. Thus, the inteins splice by a non-canonical first step. The Mma PolII intein is
regulated by the redox state of an internal disulfide bond, and the third step of splicing is inefficient with
C-terminal Gln. The Pab intein is regulated by both temperature and redox state. The thermal stability of
the unspliced Pab intein precursor allows for structural study and analysis of the kinetics of each step of
splicing in vitro. NMR solution structures of the Pab PolII intein reveal that the structure is rigid, with a
165
POSTER ABSTRACTS
disordered loop absent in an otherwise homologous P. horikoshii intein, and a b-hairpin specific to inteins
from archaebacteria. This material is based upon work supported by the National Science Foundation
under grant MCB-1244089 and the Camille and Henry Dreyfus Foundation.
POST 03-232
Directed Evolution Of A Highly Sensitive Peroxidase Reporter And Application To Electron
Microscopic Visualization Of MICU1.
Stephanie S. Lam1, Jeffrey D. Martell1, Kimberli J. Kamer2, Vamsi K. Mootha2, Alice Y. Ting1
1
Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, US, 2Systems Biology,
Harvard University, Cambridge, Massachusetts, US
Peroxidases are exceptionally versatile enzymes that have been widely utilized for biomarker detection,
fluorescence and electron microscopies, and proteomic mapping. Horseradish peroxidase (HRP) and
engineered ascorbate peroxidase (APEX) are two peroxidase reporters now available but HRP is inactive
when expressed in the cytosol of cells, while APEX lacks the exceptional sensitivity of HRP. In an effort to
close this gap, we performed yeast display evolution to engineer a novel reporter, APEX2, that is vastly
more sensitive than APEX in both living and fixed mammalian cells. The improved properties of APEX2
permitted us to examine the precise localization of MICU1, a regulator of calcium import into
mitochondria, by electron microscopy. We observed clear localization in the intermembrane space, in
contrast to previous reports of localization in the matrix, necessitating revision of a previously proposed
mechanistic model.
POST 03-233
Characterization Of The Alpha-Proteobacteria Wolbachia Pipientis Protein Disulphide Machinery
Reveals A Regulatory Mechanism Absent In Gamma-Proteobacteria
Patricia M. Walden1, Maria A. Halili1, Julia K. Archbold1, Fredrik Lindahl1, David P. Fairlie1, Kenji Inaba2,
Jennifer L. Martin1
1
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland,
Australia, 2Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai,
Aoba-ku, Japan
Dengue fever, caused by the dengue virus, is a severe and deadly disease that is still vastly expanding in
tropical and subtropical regions [1]. One way to eliminate the dengue virus is to reduce the spread of its
major vector, the mosquito Aedes aegypti. The endosymbiotic bacteria Wolbachia pipientis can
infect Aedes aegypti and inhibit dengue virus replication and transmission [2]. Because of this unique
ability of Wolbachia to interfere with the dengue virus, the bacterium has been exploited as a novel
biocontrol tool against dengue fever in tropical countries. However, the interaction
between Wolbachia and its mosquito host at a molecular level still remains poorly understood. We
hypothesize that Wolbachia secretes proteins into its host that alter physiological processes within the
mosquito. Like many other bacteria, Wolbachia possesses disulfide bond forming (Dsb) proteins that
introduce stabilizing disulfide bonds into these secreted proteins. The genome of
theWolbachia strain wMel encodes three Dsb-like proteins that could be important for stabilizing secreted
proteins: alpha-DsbA1, alpha-DsbA2, and an integral membrane protein, alpha-DsbB. Alpha-DsbA1 and
166
POSTER ABSTRACTS
alpha-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA proteins,
would need to be oxidized to the disulfide form to serve as a disulfide bond donor towards substrate
proteins. Here we show that the integral membrane protein alpha-DsbB oxidizes alpha-DsbA1, but not
alpha-DsbA2. The interaction between alpha-DsbA1 and alpha-DsbB is very specific, involving four
essential cysteines located in the two periplasmic loops of alpha-DsbB [3]. This is the first extensive
investigation of the Dsb folding machinery in Wolbachia. Exploiting Wolbachia for halting the spread of
Dengue fever is an ongoing strategy in tropical climates. References: [1] Kyle, J. L. & Harris, E. Global
spread and persistence of dengue. Annu. Rev. Microbiol. 62, 71–92 (2008) [2] Walker T, et al. (2011)
The wMel Wolbachia strain
blocks
dengue
and
invades
caged Aedes
aegypti populations. Nature 476:450–453. [3] Walden PM, Halili M, Archbold JK, Lindahl F, Fairlie D, Inaba
K, Martin JL. (2013) The alpha-proteobacteria Wolbachia pipientis protein disulfide machinery has a
regulatory mechanism absent in gamma-proteobacteria. PloS One, 8 11: e81440.1-e81440.9.
POST 03-234
The Metal-Dependent FAD Pyrophosphatase/FMN Transferase Activity Of Periplasmic FlavinTrafficking Protein (Ftp): A Potential Role In Flavoprotein Biogenesis
Ranjit K. Deka1, Chad A. Brautigam2, Wei Z. Liu1, Diana R. Tomchick2, Michael V. Norgard1
1
Microbiology, The University of Texas Southwestern Medical Center, Dallas, Texas, US, 2Biophysics, UT
Southwestern Medical Center, Dallas, Texas, US
The syphilis spirochete Treponema pallidum (Tp) is a highly enigmatic bacterium that still cannot be
cultivated in vitro, and a syphilis vaccine remains elusive. Tp lacks many biosynthetic pathways and has
evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoires. We
recently described in Tp an ABC-type riboflavin transporter (RfuABCD) that likely is essential for the
organism's survival within its human host. We also discovered treponemal Ftp (Ftp_Tp) as the first
bacterial metal-dependent FAD pyrophosphatase/FMN transferase that traffics/processes FAD into AMP
and FMN in the periplasm to generate flavoproteins for use in the redox reactions. In previous studies it
was shown thatSalmonella enterica Ftp (Ftp_Se) binds FAD, but that it lacked pyrophosphatase
activity. Although the crystal structures of these Ftps are quite similar and the FAD binding residues are
well conserved, a bi-metal catalytic center is present only in Ftp_Tp. It would thus be instructive to discern
what molecular features allow some Ftp proteins to recognize and hydrolyze FAD whereas others are
restricted to FAD binding only. Structure-based mutagenesis revealed that a single amino acid change
2+
in E. coli Ftp (Ftp_Ec) converted it from an FAD-binder to an Mg -dependent FAD pyrophosphatase
(similar to Ftp_Tp). Further, we have shown in vitro and in vivo that both types of Ftps are capable of
flavinylating proteins via the covalent attachment of FMN to the threonine sidechain of an appropriate
sequence motif. These discoveries are of widespread relevance because Ftps are essential for flavoprotein
biogenesis in the bacterial periplasm. We also propose that Tp likely possesses a flavin-based redox
system that underlies a "flavin-centric" lifestyle for Tp's survival in the human host.
POST 03-235
Characterization Of Hyperthermophilic Tyraenzymes Involved In Aromatic Amino Acid Biosynthesis
Irina Shlaifer, Joanne L. Turnbull
Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada
TyrA enzymes belong to a family of prephenate dehydrogenases that are dedicated to the biosynthesis of
the aromatic amino acid L-tyrosine. L-tyrosine is a precursor of valuable compounds such as aromatic
metabolites, pigments and hormones. Designing catalytically efficient and stable enzymes for tyrosine
overproduction in microorganisms serve as an environmentally friendly ans sustainable alternative to the
traditional methods of obtaining this amino acid from animal sources. Enzymes from hyperthermophilic
167
POSTER ABSTRACTS
organisms are of particular interest in industrial and green chemistry applications as they are stable at
neutral pH and over a wide range of temperatures.
In this study, the genes encoding putative TyrA enzymes from two hyperthermophilic archaea were cloned
and the proteins were recombinantly expressed in Escherichia coli and characterized. One is a
trifunctional enzyme from Nanoarchaeum equitans which has a prephenate dehydrogenase (PD) domain
fused to chorismate mutase (CM- the preceding enzyme in the pathway) and to the prephenate
dehydratase (PDT) that is dedicated to the biosynthesis of L-phenylalanine). The second enzyme is the
bifunctional CM-PD from ChrenarchaeonIgnicoccus hospitalis. Unlike the TyrAs from most organisms
which are mainly inhibited by the end product L-tyrosine, we have predicted by amino acid sequence
alignment that these two enzymes will be insensitive to regulation by L-tyrosine. Accordingly, kinetic
analysis of the enzymes confirms that the PDs are insensitive to L-tyrosine regulation implying that the
enzymes are good candidates for the production of industrial aromatic compounds from renewable
resources. These are the first studies exploring the aromatic amino acid biosynthetic pathway from the
two archaeal organisms which may also provide insight into the strategy adopted for the efficient
processing of labile metabolites such as chorismate and prephenate in hyperthermophilies.
POST 03-236
Probing Novel Antibiotic Targets Within Sialic Acid Catabolism
Rachel A. North2, Sarah A. Kessans2, Hironori Suzuki2, Michael D. Griffin1, Renwick C. Dobson2, 1
1
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology
Institute, University of Melbourne, Melbourne, Victoria, Australia, 2Biomolecular Interaction Centre &
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
Staphylococcus aureus is a Gram-positive bacterial pathogen, notorious for its ability to evolve resistance
mechanisms against novel antibiotics. The ever-increasing prevalence of this superbug and the continual
progression of antibiotic resistance highlight the urgency to characterize novel drug targets and develop
new drugs against this pathogen. The enzymes involved in the catabolism of sialic acids are promising
targets, yet to be exploited. Sialic acids constitute a large family of amino sugars and are undoubtedly one
of the most important carbohydrate classes in biology. Found at the terminus of glycan molecules
attached to the surface of eukaryotic cells, they are involved in a diverse range of cellular functions.
Interestingly, several bacterial pathogens capable of colonizing heavily sialylated niches can scavenge
sialic acid from their surrounding environment and degrade it as a source of carbon, nitrogen and energy.
This sequestration and subsequent catabolism of sialic acid requires a cluster of genes known as the ‘NanNag’ cluster. The Nan-Nag cluster of genes encodes five catabolic enzymes (lyase, kinase, epimerase,
deacetylase and deaminase) that degrade sialic acid via a series of reactions into fructose-6-phosphate
(see figure). This pathway has been well documented in several bacterial pathogens that colonize mucousrich niches, such as methicillin-resistant Staphylococcus aureus, suggesting that the ability to utilise sialic
acid as a ubiquitous carbon and nitrogen source is important for colonization and persistence. More
significantly, the Nan-Nag genes have proven essential for Staphylococcus aureus growth on sialic acids,
making the pathway a viable target for drug design against this pathogen. A biophysical and structural
characterization of the catabolic enzymes involved in the breakdown of sialic acid from methicillinresistant Staphylococcus aureus will be presented. Data obtained from analytical ultracentrifugation,
small-angle X-ray scattering and X-ray crystallography will be included. Crystal structures have been
obtained for N-acetylneuraminate lyase (1.7 Å), and N-acetylmannosamine-6-phosphate 2-epimerase (1.8
Å). Understanding the structural nature of these enzymes will provide us with the preliminary information
necessary for antibiotic development.
POST 03-237
168
POSTER ABSTRACTS
The Role of Protein Glycosylation in Laccases from Lentinus sp.
Wei-Chun Liu1, Manuel Maestre-Reyna2, Wen-Yih Jeng3, Cheng-Chung Lee2, Chih-An Hsu1, Tuan-Nan
Wen4, Andrew H.-J. Wang2, Lie-Fen Shyur1
1
Agricultural Biotechnology Research Center , Academia Sinica, Taipei, Taiwan, 2Institute of Biological
Chemistry, Academia Sinica, Taipei, Taiwan, 3Center for Bioscience and Biotechnology, National Cheng
Kung University, Tainan, Taiwan,4Institute of Plant and Microbial Biology, Academia Sinica, Taipei,
Taiwan
Laccases are blue multicopper oxidases catalyzed various organic and inorganic compounds by reducing
O2 to water. Despite several decades’ research, the roles of protein glycosylation in laccases are still
obscure. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4)
isolated from a white-rot fungus Lentinus sp. The nLcc4 is composed of three cupredoxin-like domains
D1-D3, and each domain folds into a Greek key β-barrel topology. The T1 and T2/T3 copper binding sites
75
238
458
and three N-glycosylated sites at Asn , Asn , and Asn were elucidated. Initial rate kinetic analysis
-1
-1
revealed that the specific activity, kcat, Km, and kcat/Km of nLcc4 with ABTS were 2,996 Umg , 3,412 s , 65.0
-1
-1
± 6.5 μM, and 52 s μM , respectively, and the values for lignosulfonic acid determined using isothermal
-1
-1
-1
-1
titration calorimetry was 39 Umg , 44 s , 42.0 ± 2.1 mM, and 1.05 s mM , respectively. Endo Hdeglycosylated nLcc4 (dLcc4), leaving one GlcNAc residue at the three N-glycosylated sites, exhibited
similar kinetic efficiency and thermal stability to that of native nLcc4. However, the N75D, N238D and
N458D mutant enzymes secreted in the cultural media of Pichia pastoris cells maintained only 4-50%
activity of the wild-type laccase. Molecular dynamics simulations analysis of various states of (de)glycosylation in nLcc suggest that the local H-bonds networks between domain connecting loop D2-D3
and glycan moiety play a crucial role in laccase activity. Together, this study provides new insights in the
structural and functional roles of glycosylation in Basidiomycetes fungal laccase.
POST 03-238
FBP17 Plays A Role In The Morphological Control By Regulating The Activity Of Rho Subfamily
GTPase CDC42
Jun Zhang, Lin Ming-ming, Zhang Qian-ying, Wang Yun-hong, Li Xin
Institute of Molecular Medicine and Oncology, Chongqing Medical University, Chongqing, China
The formin-binding protein 17 (FBP17) is widely expressed in eukaryotic cells and found to be involved
with endocytosis and migration in macrophage. In these cellular processes, the N-terminal EFC (extended
FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was
previously shown to have membrane binding and deformation activities. In hepatocyte, it was
interestingly found that knockdown of the endogenous FBP17 by RNAi didn’t affect the cell endocytosis
and migration, but leaded to the cell morphological transition from the epithelioid to fibroid. Further
investigation demonstrates that the activity of CDC42, which is governed by the exchange of GTP and
GDP, significantly reduces with the down-regulation of FBP17 and withdrawal of the silence could
simultaneously recover the FBP17 expression, the activity of CDC42 and the cell morphology. Polypeptide
specifically targeted to the RBD domain of FBP17, a putative Rho family protein binding site, can also
attenuate the CDC42 activity with a similar morphological response, which strongly suggests that FBP17
might play an important role in morphological control to keep moderate CDC42 activity possibly by the
physical interaction of its RBD domain with CDC42. Co-localization of RBD domain of FBP17 and CDC42
has been directly observed in the cell cortex. Furthermore, F-actin stabilizing reagent phalloidine can
abrogate the morphological responses mediated by both the FBP17-siRNA and target polypeptide, which
implies that the inhibition of CDC42 activity by knockdown of FBP17 initially results in disrupting the
equilibrium of F-actin dynamics, followed by rearrangement of actin cytoskeleton in cortex and
subsequently forces the morphological remodeling. It is indicated that FBP17 could be responsible for the
169
POSTER ABSTRACTS
versatile functions in different cellular milieus via distinct molecular pathways. Key words: FBP17;
morphological remodeling; CDC42; cell cortex; actin dynamics This work is funded by the grants from
Chongqing Education Commission (KJ080301), Chongqing Science&Technology Commission (CSTC,
2010BB5366) and National Natural Science Foundation of China (NO. 20803098).
POST 03-239
Semi-Synthesis and Applications of Fluorophore/Thioamide pairs Containing Proteins
Solongo Batjargal, E. James Petersson
University of Pennsylvania, Philadelphia, Pennsylvania, US
Our laboratory is interested in elucidating the structures of misfolded proteins on the single residue level
using fluorescence spectroscopy. We have shown that a thioamide, a substitution of the carbonyl oxygen
with a sulfur atom in the peptide backbone, is capable of quenching a variety of fluorophores. We have
developed methods for incorporating fluorophore/thioamide pairs into full-length proteins through a
combination of unnatural amino acid (Uaa) mutagenesis and native chemical ligation (NCL). We have
applied our small thioamide probes to studies of α-Synuclein (αS), an intrinsically disordered protein
implicated in Parkinson’s disease. The conformational changes of the αS monomers labeled with
fluorescein/thioamide pairs have been examined in the presence of osmolytes as well as in aggregation
prone states through fluorescence spectroscopy. In addition, we have developed an easy and efficient
method of isolating the full-length proteins containing Uaa insertions by exploiting the unique selfsplicing properties of inteins. A C-terminal His-tagged intein provides a convenient handle to pull out
only the full-length protein, and the subsequent removal of the intein in a traceless manner yields the
Uaa-labeled protein in pure form. This technique should be of general use to the Uaa mutagenesis
community as it provides a simple solution to the “truncation problem.”
POST 03-240
Computational Design of an Unnatural Amino Acid Dependent Metalloprotein with Atomic Level
Accuracy
Jeremy Mills1, Sagar D. Khare1, Jill M. Bolduc2, Farhad Forouhar3, Vikram K. Mulligan1, Scott Lew3,
Jayaraman Seetharaman3, Liang Tong3, Barry L. Stoddard2, David Baker1
1
Biochemistry, University of Washington, Seattle, Washington, US, 2Division of Basic Sciences, Fred
Hutchinson Cancer Research Center, Seattle, Washington, US, 3Biological Sciences, Columbia University,
New York, New York, US
Genetically encoded unnatural amino acids (UAAs) could provide a foundation for the development of
proteins with novel chemistries or functions. However, as success in this endeavor requires precise
placement of the UAA and surrounding residues within a protein scaffold, it represents a difficult
challenge. Computational protein design methods could potentially be employed to address this problem,
but had not been used in concert with UAAs prior to this work. We report the use of the Rosetta
computational protein design suite to engineer a novel metalloprotein in which the UAA (2,2′bipyridin5yl)alanine (Bpy-ala) serves as a primary ligand of the bound metal. In a first design attempt,
important considerations were identified for design approaches that utilize Bpy-ala as a metal ligand. A
second round of design identified a series of candidate proteins in which octahedral binding sites were
formed from Bpy-ala, two scaffold based metal liganding residues, and two water
molecules. Experimental characterization of these candidates identified MB_07 as a protein possessing
2+
2+
2+
2+
the ability to bind with high affinity a host of divalent cations including Co , Zn , Fe , and Ni . X-ray
2+
2+
crystallographic characterization of MB_07 bound to Co and Ni showed excellent agreement between
the computational models and the designed metalloprotein; RMSDs over all atoms in the binding site are
2+
0.9, and 1.0 Å respectively. The dissociation constant of MB_07 for Zn was measured to be ~40 pM,
170
POSTER ABSTRACTS
5
representing a ~2x10 increase in affinity of the designed protein for this metal relative to the UAA alone.
The methodologies developed in the course of this study should facilitate the design of novel proteins
with properties that would be difficult to achieve exclusively using naturally occurring amino acids.
POST 03-241
Acrolein-modified High Density Lipoproteins Promote Atherogenesis
Alexandra Chadwick1, Rebecca L. Holme1, Yiliang Chen2, Kirkwood A. Pritchard1, Daisy Sahoo1
1
Medical College of Wisconsin, Milwaukee, Wisconsin, US, 2Blood Research Insititute, Blood Center of
Wisconsin, Milwaukee, Wisconsin, US
High density lipoprotein (HDL) prevents atherosclerosis through its key roles in the reverse cholesterol
transport pathway (RCT), where HDL’s main function is to transport excess cholesterol from the periphery
to the liver for excretion. Cholesterol delivery into hepatic cells is achieved by interaction of HDL with its
receptor, scavenger receptor BI (SR-BI). Mounting evidence shows that modification to HDL by
environmental factors may reduce/eliminate its anti-atherogenic functions. Acrolein, a highly reactive
aldehyde found in tobacco smoke, has recently been identified as an HDL modifier in vivo, although its
affects on HDL function in RCT have not been fully characterized. We hypothesized that acrolein
modifications to HDL (acro-HDL) generate a dysfunctional particle that: (i) is unable to mediate SR-BImediated selective uptake of HDL-CE and efflux of free cholesterol (FC) and (ii) upregulates inflammatory
pathways in macrophages that promote atherogenesis. To test this hypothesis, human HDL was first
modified with 250uM acrolein. Immunoblot analysis confirmed the presence of protein crosslinking after
one hour of acrolein modification, as well as the presence of acrolein adducts on apoA-I and apoA-II, the
two major HDL proteins. Mass spectrometry analyses also verified the expected acrolein adduct shifts of
56, 76, and 94 Da. Next, we assessed acro-HDL’s functionality during the beginning of RCT. Our data
revealed that acro-HDL displayed a 30% decrease in accepting FC effluxed out of SR-BI-transfected COS7
cells, as compared to native HDL. Oil Red O staining of murine macrophages supported these findings.
We then evaluated acro-HDL’s functionality at the end of RCT and demonstrated that acro-HDL (050mg/mL) was 10-25% less efficient at delivering HDL-CE to COS7 cells transiently expressing SR-BI.
Finally, we used qRT-PCR to demonstrate that acro-HDL, but not native HDL, stimulated atherogenic
pathways by increasing mRNA expression of scavenger receptors (CD36 and LOX-1) and inflammatory
markers (MCP-1, IL-8, ICAM-1, and TNF-a) in human THP-1 differentiated macrophages, paralleling the
effects of oxidized LDL . Altogether, our findings reveal that acrolein modification of HDL produces a
dysfunctional particle that is proatherogenic. More detailed investigations into how oxidative modification
of HDL contributes to atherosclerosis will be extremely important in combating cardiovascular disease.
POST 03-242
Control Of Protein Production And Virus Replication By Pharmacological Blockade Of Degron
Detachment
Hokyung K. Chung1, Conor Jacobs1, Yunwen Huo6, Jin Yang5, Stefanie A. Krumm4, Richard K. Plemper4, 7,
Rodger Y. Tsien5, 2, Michael Z. Lin6, 3
1
Department of Biology, Stanford University, Stanford, California, US, 2Department of Chemistry and
Biochemistry and Howard Hughes Medical Institute, University of California, San Diego, La Jolla,
California, US, 3Department of Pediatrics, Stanford University, Stanford, California, US, 4Department of
Pediatrics, Emory University, Atlanta, Georgia, US, 5Department of Pharmacology, University of
California, San Diego, La Jolla, California, US,6Department of Bioengineering, Stanford University,
Stanford, California, US, 7Institution for Biomedical Science, Georgia State University, Atlanta, Georgia,
US
171
POSTER ABSTRACTS
A generalizable method to rapidly control protein production while minimizing protein modification is
desirable for many biological applications. Here, we describe a novel method for pharmacological shut-off
of protein production using an essentially traceless protein tag. This tag is genetically encoded to the
protein of interest (POI) and consists of the self-cleaving HCV protease and degron. By default, this tag is
separated from the POI and leaves it structurally unmodified; however, administration of a clinically
approved HCV protease inhibitor blocks degron detachment on the subsequently synthesized POI. The
POI fused to the degron will then be proteolytically removed. We call this system, Small-Molecule Assisted
Shutoff (SMASh). The traceless characteristic of SMASh is especially beneficial for controlling a wide range
of proteins whose structures need to be intact for their functions such as virus replication factors. We
illustrated the usefulness and versatility of SMASh by showing reversible control of various proteins’
production in multiple mammalian cell lines. Importantly, SMASh enables us to engineer drugsusceptibility into the measles virus, an oncolytic RNA virus that is in clinical trials but does not have a
specific inhibitor. This drug-regulable SMASh-incorporated virus would hold potential as a safe platform
virus in translational applications such as cancer therapy or new multivalent live vaccine. Together with the
advantages of the commercial HCV inhibitor–non-toxicity, high selectivity and cell-permeability– we
envision that the rapid, reversible, uniquely traceless and compact nature of SMASh will allow a variety of
applications in biomedical research and technology.
POST 03-243
Understanding The Metabolism Of Enteric Pathogen Campylobacter jejuni
Adnan Ayna, Peter Moody
Leicester University, Leicester, United Kingdom
Fructose bisphosphatase (EC 3.1.3.11) is the enzyme that converts fructose-1,6-bisphosphate to fructose
6-phosphate in gluconeogenesis. In mammals the enzyme is subject to metabolic regulation but
regulatory enzymes of bacterial FBPases are relatively less understood. Our study aimed to shed some
lights on the metabolism of enteric pathogen Campylobacter jejuni. Homology modeling and sequence
alignments show that the FBPase from C. jejuni (CJFBPase) is expected to have the same overall fold as the
other type 1 FBPases for which structural information is available. However, alignments of the sequences
show different trends in AMP, Glc-6-P and PEP binding sites. Some of the critical residues of AMP binding
sites may be missing in C. jejuni and some seem not to be conserved. The regulatory properties of the
enzymes have been investigated and surprisingly it is found that physiological level of ATP has an
inhibitory effect on the activity of CJFBPase suggesting that there might be a novel ATP binding site on
the enzyme. PEP also has an inhibitory effect on CJFBPase. It is not clear how PEP inhibits the activity as it
doesn’t have the conserved PEP binding site. Both molecules are uncompetitive inhibitors of the enzyme
as they decrease Km and Vmax. To test the effect of these molecules on the thermal stability of the
enzymes the effect of thermal variation on the stability was investigated with CD using 1 mM PEP and 2.5
mM ATP. Both molecules increased the stability of the enzyme supporting the suggestion that there
might a novel ATP binding site of the enzyme. In order to fully understand the mechanism of inhibition,
crystal structure of the enzyme needs to be solved.
POST 03-244
Chitosan-Binding Modules (CBM32) of a chitosanse From Paenbacillus sp. IK-5 ---Amino Acid
Residues Responsible For Chitosan Binding---
Shoko Shinya3, Takayuki Ohnuma3, Hisashi Kimoto2, Hideo Kusaoke1, Tamo Fukamizo3
1
Fukui University of Technology, Fukui, Japan, 2Fukui Prefecture University, Fukui, Japan, 3Kinki
university, Nara-shi, Japan
172
POSTER ABSTRACTS
A chitosanase isolated from Paenibacillus sp. IK-5 has two carbohydrate binding modules (DD1 and DD2)
at its C-terminus (Kimoto et al., J. Biol. Chem. 2002, 277, 14695-14702). DD1 and DD2 are highly specific to
chitosan oligosaccharides, (GlcN)n. Although the amino acid sequence of DD1 is highly homologous (72
%) to that of DD2, the affinity of DD1 to (GlcN)n is much higher than that of DD2. NMR titration
experiments and docking simulations showed that in both DD1 and DD2, (GlcN)nbind to the shallow
binding cleft formed by several loops extruded from the b-sandwich structure (Shinya et al., J. Biol.
Chem. 2013, 288, 30042-30053). However, we found that Glu36 located in the (GlcN)n-binding site of DD1
is substituted with tyrosine in DD2, as shown in Fig. 1. To identify the structural factors resulting in the
different affinities between DD1 and DD2, Glu36 of DD1 was mutated to tyrosine (DD1-E36Y), and the
reverse mutation (Tyr→Glu) was also conducted at the 36thposition in DD2 (DD2-Y36E). For the NMR
titration experiments, 15N- and 13C-labeled DD1, DD1-E36Y, DD2, and DD2-Y36E were obtained using E.
coli expression system in an M9 minimal medium containing 15N-NH4Cl and 13C-glucose. Based on the
chemical shift perturbations of the 1H-15N HSQC resonances, (GlcN)4 binding ability was evaluated for
individual module proteins. The resonances of amino acids located at the (GlcN)n binding site of DD1
were affected strongly, whereas the resonances of DD1-E36Y were almost unaffected. In contrast, the
resonances of DD2-Y36E were more strongly affected than those of DD2. ITC experiments were also
conducted to determine the association constants (Ka) of (GlcN)4 for individual proteins. The Ka values for
(GlcN)4 were 4.7x 105 M-1 for DD1 and 5.6 x 103 M-1 for DD1-E36Y. The mutation of Glu36 of DD1
significantly reduced the binding affinity. The Ka values were 5.8 x 103 M-1 for DD2 and 3.1 x 104 M-1 for
DD2-Y36E. The mutation of Tyr36 of DD2 enhanced the binding affinity. Thus, we concluded that amino
acid substitutions at the 36th position significantly control the binding ability of DD1 and DD2.
POST 03-245
Crystal Structure Of A Family GH18 (Class V) Chitinase From Cycad, Cycas revolta---- Structural
Factors Controling The Transglycosylation Activity Of The Enzyme
Tamo Fukamizo1, Naoyuki Umemoto1, Takayuki Ohnuma1, Toki Taira2, Tomoyuki Numata3
1
Department of Advanced Bioscience, Kinki University, Nara, Japan, 2Department of Bioscience and
Biotechnology, University of Ryukyus, Nishihara, Okinawa, Japan, 3Biomedical Research Institute,
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
A family GH18 chitinase from cycad, Cycas revolta, (CrChiA) is the first plant chitinase that has been found
to possess transglycosylation (TG) activity. To identify the structural factors controlling the TG activity, we
solved the crystal structure of the chitinase, and compared the structure with those of GH18 chitinases
from Arabidopsis thaliana (AtChiC) and Nicotiana tabaccum (NtChiV), which do not exhibit TG activity, All
three chitinases have an (α/β)8 barrel fold with an insertion domain (green region), as shown in Fig. 1. In
the acceptor-binding site (+1, +2, and +3) of CrChiA, several aromatic side chains form a cluster,
stabilizing the platform for the oligosaccharide binding. Among the aromatic side chains in the cluster,
Trp168 was found to make a face-to-face stacking interaction with the +3 sugar. However, the cluster in
the identical region of AtChiC is smaller than that of CrChiA. Tryptophan residue is not conserved in this
cluster of AtChiC. Such a cluster was not found in the identical region of NtChiV. This situation may bring
about the lower affinity for acceptor binding to AtChiC and NtChiV. In the catalytic DxDxE motif (the
bottom of Fig. 1), the side chain of the middle Asp (Asp117) is always oriented toward Glu119 in CrChiA,
whereas in NtChiV and AtChiC, the side chain of the middle Asp (Asp113 and Asp114, respectively) is
oriented toward the first Asp (Asp111 and Asp112, respectively). The orientation of the middle Asp toward
Glu in the DxDxE catalytic motif was also found in the Serratia chitinase (SmChiA) mutant (W167A), and
suggested to be one of the molecular signs for efficient TG activity in GH18 enzymes. CrChiA, NtChiV, and
AtChiC were also employed for allosamidin binding experiments using isothermal titration calorimetry. No
significant difference in the free energy changes of allosamidin binding (ΔG○) to subsites -3, -2, and -1
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POSTER ABSTRACTS
was found for these chitinases; -9.7, -9.8, and -9.5 kcal/mol for CrChiA, NtChiV, and AtChiC, respectively.
However, the solvation and conformational entropy changes (ΔSsolv and ΔSconf) for CrChiA were
considerably different from those for NtChiV and AtChiC but simillar to those for SmChiA, which catalyzes
TG reaction. Taken together, structural factors controlling the TG activity were found to lie on the entire
region of the binding cleft; the acceptor-binding site, the catalytic center, and the allosamidin binding
site.
POST 03-246
Split Intein Mediated Peptide Cyclization
Shubhendu Palei2, 1, 3, Henning D. Mootz2, 1, 3
1
Department of Chemistry and Pharmacy, Muenster, North Rhine Westphalia, Germany, 2Institute of
Biochemistry, Muenster, Germany, 3International NRW Graduate School of Chemistry, Muenster,
Germany
Cyclic peptides are in the focus for therapeutic applications because of their unique properties. Peptide
backbone cyclization is one of the major strategies to stabilize peptides and proteins. Cyclic peptides are
more stable towards pH change, heat denaturation, protease degradation than their linear counterparts.
The cyclic backbone also has less conformational flexibility and small entropy which increase their binding
affinity towards the peptide or protein interface. Therefore cyclic peptides are also used as inhibitors for
protein-protein interactions or for different enzymes. There are different methods for achieving cyclization
of peptide backbone either by chemical or biological means. Protein trans-splicing (PTS) is one of the
robust ways of making cyclic backbone in vitro as well as in vivo. In PTS, the extein sequences from two
different protein and/or peptide partners are joined by a new peptide bond without the use of any
external energy or cofactor. It is purely an autocatalytic process. Here we develop a novel method of
peptide backbone cyclization using PTS to make a range of cyclic peptides of different sizes. We make the
use of SspDnaB split intein to make cyclic peptides. This technique can be used as an excellent tool to
make cyclic peptides even in complex environments in vitro (cell lysates) or in vivo (inside a living cell)
with a great selectivity.
POST 03-247
Exploring the Morpheein Forms of B. cenocepacia HMG-CoA Reductase
Riley Peacock, Michelle Brajcich, Courtney Boyd, Jeffrey Watson
Chemistry and Biochemistry, Gonzaga University, Spokane, Washington, US
Isoprenoids are a key class of biological molecules required by all three kingdoms of life, and are
synthesized by one of two metabolic pathways. The mevalonate pathway is found in eukaryotes, archaea
and some eubacteria, and proceeds through the enzyme HMG-CoA reductase (HMGR), which catalyzes
the rate-limiting and first committed step. The DXP pathway is found in most eubacteria, and does not
use HMGR at any step. The opportunistic lung pathogen Burkholderia cenocepaciacodes for HMGR, yet
biosynthesizes isoprenoids using the DXP pathway. B. cenocepacia is a significant cause of fatality in
cystic fibrosis patients, in part due to its natural resistance to most available antibiotics. Therefore,
understanding the function and regulation of HMGR from B. cenocepacia (BcHMGR) may aid in
combating these difficult-to-treat infections. Our evidence from various kinetic, spectroscopic and
chromatographic techniques suggests that BcHMGR exists in at least three active quaternary forms, each
with distinct size and charge properties. In addition, our data suggests that the equilibrium between
these states depends on pH, enzyme concentration, and substrate concentration, as changing these
elements independently yields a shift in the relative concentration of these forms, and an associated
change in the kinetic activity of the enzyme. This data suggests that BcHMGR follows the morpheein
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POSTER ABSTRACTS
model of allostery, and further suggests that the enzyme may be an important asset to B.
cenocepacia and a potential antibiotic target.
POST 03-248
Dynamic Functional Switch in Poliovirus 3C Protease
Yan M. Chan, David D. Boehr
Chemistry, Pennsylvania State University, University Park, Pennsylvania, US
Viral genomes are very efficient; they are typically compact but nevertheless encodes numerous elements
that are essential for regulation of both its own replication and packaging, and of the host cell's
machinery. Viruses have developed intelligent strategies to overcome their biological information storage
pro
problem. For example, the 3C protein from the picornavirus family of positive-strand RNA viruses is
responsible for binding of RNA control sequences to regulate translation and replication, interacting with
phosphoinositide lipids (PI) to regulate the maturation of virus replication organelles, and acting as the
pro
main protease to cleave host and virus proteins to further regulate host and virus processes. 3C can also
pro
pro
pro
be found as a domain in the 3CD polyprotein. 3C by itself and 3CD have different protease
pro
specificities, and likely different RNA and PI binding capabilities. The domains in 3CD are tethered by a
pro
pol
flexible linker and do not make specific 3C -3D interactions. Surprisingly, we have found that by
extending the C-terminal tail of 3C with just a few amino acid residues, the RNA and PI binding properties
alter dramatically. These functional changes are accompanied by changes in the structural dynamics of 3C,
as measured by NMR relaxation methods. We propose that this finding has critical bearing on 3C
pro
pro
function; proteolytic processing of the C-terminus is the switch from 3CD to 3C (by itself) activities.
Such a simple, but elegant, mechanism does not require any additional domain-domain interactions in the
pro
pro
3CD polyprotein to regulate 3C function, and can help explain functional differences between
pro
pro
3C and 3CD that have confounded virologists and structural biologists for years.
POST 03-249
Lighting the Cellular Fuel Gauge with Fluorescent Sensors for Imaging Single-Cell Metabolism
Mathew Tantama, Juan Ramón Martínez-François, Rebecca Mongeon, Gary Yellen
Neurobiology, Harvard Medical School, Boston, Massachusetts, US
How do neurons support their metabolically-demanding signaling activities? Despite our growing
knowledge of brain circuitry, we still do not understand how neurons and glia tune their cellular
metabolism to support different types of signaling. In order to interrogate this linkage between
metabolism and brain activity, genetically-encoded biosensors have been developed to visualize cellular
energy status using live-cell optical microscopy. In particular, a fluorescent biosensor, PercevalHR, has
now been optimized to sense the range of intracellular ATP:ADP ratios expected in mammalian cells and
to respond to changes within seconds. Additionally, this newly engineered biosensor can be used with
either one- or two-photon fluorescence excitation microscopy with live samples. To demonstrate that it is
a sensitive reporter of physiological changes in energy consumption and production, PercevalHR has been
used to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple
stimuli. PercevalHR has also been used to visualize intracellular ATP:ADP while simultaneously recording
electrical currents from ATP-sensitive potassium (KATP) channels, enabling the direct correlation between
cytosolic ATP:ADP and KATP channel open probability in intact single cells. Thus, PercevalHR should be a
versatile tool that will provide access to a new level of molecular and cellular detail in the study of energy
metabolism. Furthermore, this combination of genetically-encoded biosensors, live-cell imaging, and
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POSTER ABSTRACTS
electrophysiology will be used to study the metabolic components of neurodegeneration in aging and in
diseases such as epilepsy and Parkinson's.
POST 03-250
Structural Analysis And Molecular Dynamics Of The Self-Sufficient P450 CYP102A5 And CYP102A1:
A Combined Computational/Experimental Approach To Increase The Efficiency Of Biocatalyst
Engineering
Maximilian Ebert4, 1, Brahm Yachnin2, Guillaume Lamoureux3, 1, Albert Berghuis2, 1, Joelle Pelletier4, 1
1
PROTEO, Montreal, Quebec, Canada, 2McGill University, Montreal, Quebec, Canada, 3Concordia
University, Montreal, Quebec, Canada, 4Université de Montréal, Montreal, Quebec, Canada
P450s catalyze the oxidation of non-activated carbon atoms, which is chemically demanding. Members of
the CYP102 family are termed “self-sufficient P450s”, meaning that they contain all the machinery
necessary to ensure the electron transfer and active site regeneration in one single protein. However, the
macromolecular assembly remains unknown. Here we report results of SAXS analysis that bring new
insights into the formation of the active complex. The recently reported new member CYP102A5 is highly
interesting due to its sequence similarity to the intensively studied member CYP102A1, accompanied by a
significant increase in electron transfer rate and improved regioselectivity. Homology models were
generated, compared and used to identify the structural basis for these differences in catalytic activity.
Based on this result we identified residues that may be involved in the gating and substrate capturing
mechanism in CYP102A5. Predictions of differences in substrate incorporation and product release from
the active site were computed using the adaptive biasing force (ABF) method. With this pioneering
application of ABF in enzyme engineering, we were able to predict all known important residues for fatty
acid substrate binding in CYP102A1, as well as two additional residues which were identified and
analyzed in vitro to support the in silico finding. This newly developed computational biology approach, in
addition to conformational studies, will help to guide directed evolution efforts towards the oxidation of
non-native substrates.
POST 03-251
Isolation And Characterization Of Proline Specific Dipeptidyl Peptidase IV From The Tenebrio
Molitor Larval Midgut
Valeriya F. Sharikova1, Irina Goptar1, Yulia Smirnova2, Brenda Oppert3, Irina Filippova1, Elena Elpidina2
1
Chemistry, Lomonosov Moscow State University, Moscow, Moscow, Russian Federation, 2A.N.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian
Federation, 3Center for Grain and Animal Health Research, Stored Product Insect Research Unit,
Manhattan, Kansas, US
The yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) is a stored product pest and a
biochemical model organism of the family Tenebrionidae. The main food proteins of this insect are
storage seed proteins - prolamins, which are also present in the diet of most people. Prolamins contain 10
- 30% proline and 30 - 50% glutamine residues. Several proline- and glutamine-rich prolamin peptides,
which are resistant to proteolysis by human digestive enzymes, cause autoimmune Celiac Disease in 1% of
the susceptible human population. In this regard, proline specific peptidases (PSP) are urgently needed for
therapeutics, capable of hydrolyzing peptide bonds formed by proline residues that are resistant to
proteolysis by peptidases of broad substrate specificity. We have carried out a bioinformatic search for
PSP in the T. molitor larvae gut transcriptome and found 12 sequences similar to human PSP. Two
predicted dipeptidyl peptidase IV (DPP IV) sequences were found, one with the highest mRNA expression
level among all PSPs in the larval transcriptome. These enzymes are serine exopeptidases from the S9
family, which cleave Xaa-Pro dipeptides from the N-terminus of polypeptides. The major DPP IV-like
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POSTER ABSTRACTS
enzyme was purified from the T. molitor larvae midgut and analyzed by mass-spectrometry analysis. The
amino acid sequence of the enzyme coincided with that of the highly-expressed DPP IV found
in T. molitor gut transcriptome. The isolated enzyme was characterized by substrate specificity, pHdependence, pH stability, and inhibitor sensitivity. The importance of DPP IV in insect digestion as well as
the potential for new treatments of Celiac Disease will be discussed. This work was supported by ISTC
grant 3455 and RFBR grants 12-03-01057-a, 14-04-91167-NSFC_a.
POST 03-252
Characterization Of Hydrolytic Enzyme-Producing Bacteria Isolated From Paper Mill
Manel Ghribi5, 2, Fatma Meddeb-Mouelhi2, 3, Marc Beauregard1, 2, 4
1
biologie médicale, Université de Québec à Trois Rivieres, Trois-Rivieres, Quebec, Canada, 2CRML, TroisRivieres, Quebec, Canada, 3Buckman North America, Vaudreuil-Dorion, Quebec, Canada, 4PROTÉO,
Laval, Quebec, Canada, 5physique,chimie et biochimie, Université de Québec à Trois-Rivières, TroisRivieres, Quebec, Canada
Enzymes act as biocatalyst in many industries, such as textiles, detergent, food, animal feed, bio-fuel,
paper and pulp, pharmaceutical, to name a few. Cellulases and hemicellulases are efficient hydrolytic
enzymes used in the pulp and paper industry to reduce the cost of production. In addition, industrial
enzymes reduce the environmental impact by replacing harmful chemicals. The leading industrial enzyme
suppliers (Novozymes, Genencor) offer a limited library of enzymes. Thoses enzymes are poorly adapted
to the need of the paper industry. Therefore, our partner, Buckman North America, wants to expand his
enzymes portfolio by characterizing new enzymes-producing bacteria. Pulp and paper mills offer
untapped biodiversity for microorganisms that use cellulose-based substrates as nutriments. In this
project, we will isolate and characterize cellulose and hemicellulose degrading microorganisms from
paper mill sludges. To conserve microorganism’s biodiversity, we used two temperatures (37 ᵒC and 50 ᵒC)
for the isolation step. Detection of extracellular enzymatic activities was carried out on minimum agar
plate medium supplemented with either cellulose (carboxylmethylcellulose or Avicel) or beechwood
xylans. Bacteria strains showing extracellular cellulase and/or xylanase activities were isolated from various
sludges (primary, secondary, presses and machines) found in a paper mill. These bacteria were identified
based on their morphology, biochemical characterization and DNA 16s sequencing. The biorefining
potential of these enzymes will be evaluated.
POST 03-253
Isolation of NRPS and PKS Gene Clusters from Soil Microbes
Danielle N. O'Hara, Connor P. Craig
Greater than 90% of microbes living in soil are unculturable due to their complex nutrient and
temperature requirements for growth. These microorganisms present a potential source of natural
products that could be developed for biotechnological and pharmaceutical uses. Microorganisms with
phosphopantetheinyl transferase (PPTase) activity are of high interest due to the role PPTase plays in
activating non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene cluster
products. The proteins expressed by these gene clusters synthesize complex natural products that are
utilized by the microorganism or its host for selective advantages. We screened for NRPS and PKS gene
clusters in microbes isolated from soil on the University of Richmond campus. Genomic DNA was isolated
from each of these samples and was used to construct metagenomic libraries. We will continue to screen
the libraries for PPTase activity in order to identify positive clones. We extended this study to include a
library of pigmented microbes previously isolated from Chesapeake Bay sponges, Clathria
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POSTER ABSTRACTS
prolifera and Halichondria bowerbanki. The library was screened for antibacterial activity using gram
positive and Gram negative co-cultures.
POST 03-254
Treatment Of Kraft Pulp With Enzymes For Improving Beatability And Physical Properties
Li Cui1, 2, Fatma Meddeb1, 3, Marc Beauregard1, 2
1
CRML, Université du Québec à Trois-Rivières, Trois-Rivieres, Quebec, Canada, 2PROTEO, Université
Laval, Québec, Quebec, Canada, 3Buckman North America, Vaudreuil-Dorion, Quebec, Canada
Over the last few years, the importance of enzymes as biotechnological catalysts for the pulp and paper
industry has been demonstrated. Among the enzymes mostly studied, hydrolases such as cellulases and
hemicellulases have been investigated for their potential impact on refining energy costs. Unfortunately,
energy reduction by prior enzyme treatments often had a negative impact on certain paper properties.
In this study, five different commercial cellulase formulations were used for the modification of fiber
properties. PFI refining was employed at 3000 and 4500 revolutions to mimic the impact of various
levels of refining, particularly on fiber size and morphology. With the five enzyme preparations, it was
possible to decrease the number of PFI revolutions by 50% and achieve the same target freeness value
(decrease in pulp CSF by approximately 200 mL) afforded by more intense refining without enzyme. The
fiber morphology changed to different extent according to various enzymatic treatments. Subsequently,
the carbohydrates in the filtrate released during enzymatic treatment was studied by ion
chromatography (IC). IC results showed good agreement with the enzyme activity measured
independently. The impact of enzymatic treatment on physical properties of handsheet was also
investigated. The enzyme impact on tear index was exceptional compared to most properties measured
in this study. Enzymes had a deleterious impact on tear even without any mechanical refining. A slight
decrease in tear strength was observed with enzyme C1 and C4 at pH 7 after mechanical refining (less
than 10%) while the most important decrease in tear was observed after C2, C3, C5 treatments. The
reason for this phenomenon appears to be that C1 and C4 had xylanase activity. We conclude that
xylanase activity could preserve and/or improve the properties of handsheets made from enzymatic
treated pulp, and that the balance between cellulolytic and hemicellulolytic activities is the key to
optimization of biorefining, leading to energy reduction and improving handsheet properties.
POST 03-255
Structural And Biochemical Investigation Of The Intramolecular Interactions Of Ceramide Transfer
Protein
Jennifer Prashek, Seungkyung Kim, Xiaolan Yao
University of Missouri-Kansas City, Kansas City, Kansas, US
Ceramide transfer (CERT) protein transfers ceramide, a key intermediate in sphingolipid biosynthesis, from
the endoplasmic reticulum to the Golgi where it is converted to sphingomyelin. CERT contains several
domains and motifs. The amino terminal pleckstrin homology (PH) domain targets CERT to the Golgi by
specifically recognizing phosphatidylinositol-4-phosphate (PtdIns(4)P). The carboxyl terminal
steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain catalyzes the transfer
of ceramide. Following the PH domain is a short stretch rich in serine and threonine residues called the
Serine Rich (SR) motif. Phosphorylation of the SR motif reduces the ceramide transfer activity of CERT. This
is achieved through both inhibition of the PH domain binding to PtdIns(4)P and START domain transfer of
ceramide. It has been shown that this inhibition requires both the PH and START domains suggesting an
1,2
auto-inhibitory intramolecular interaction . We set out to investigate the structural basis of
phosphorylation inhibition of CERT function. We generated mutations of ten serine and threonine
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POSTER ABSTRACTS
residues of the SR motif to glutamate to mimic the phosphorylated form of CERT. We also generated
isolated PH and START domains to further investigate the domain-domain interaction within CERT using a
combination of structural and biochemical methods. Our results show that the interaction of the PH and
START domains may contribute to the inhibition of PH binding to PtdIns(4)P that occurs upon SR motif
phosphorylation. We are currently investigating the effect of disrupting the PH-START interaction on the
activity of the phosphorylation mimic of full length CERT.
POST 03-256
Evaluating Interpretation Of B-factors For Collective Motion Modeling
Edvin Fuglebakk1, Nathalie Reuter1, Konrad Hinsen2
1
University of Bergen, Bergen, Norway, 2Centre de Biophysique Molećulaire, Centre Nationalde la
Recherche Scientifique, Orleáns, France
The interpretation of crystallographic B-factors in terms of thermal motion is prevalent in many
branches of protein science. It is therefore important to understand its inherent limitations. One
common criticism against interpreting functional motion from B-factors is that they are influenced by
many non-thermal factors. Another concern is that the thermal component of B-factors arises from
motion in a highly restrictive crystalline environment. This environment is expected to dampen
collective motion, the kind of motion often involved in functional rearrangments like conformational
change. I will present results that reveal potential problems with the use of B-factors as a model of
thermal motion of solvated proteins. We have compared the collective motions of several elastic
network models, a kind of protein model commonly validated and parameterized against B-factors. We
obtained collective motion predictions from elastic network models and compared them with molecular
dynamics simulations for seven solvated protein structures. We find that models that give good
reproduction of B-factors are severely compromised in their ability to recapitulate collective motions.
Moreover, we compare the elastic network models with a null model of restricted collective motion, and
find that models parameterized to reproduce B-factors are in close agreement with this null model. We
therefore find it important to consider the effect of the crystalline environment when interpreting Bfactors, and avoid inference about slow collective motion from this data.
POST 03-257
Structure/Function Relationships in Carboxylesterase EstGtA2 from Geobacillus thermodenitrificans
Jessica K. Moisan1, 2, Fatma Meddeb-Mouelhi1, 3, Marc Beauregard1, 4
1
3351 Boulevard des Forges, Université du Québec à Trois-Rivières, Trois-Rivières, Quebec,
Canada, 2PROTEO, Québec, Quebec, Canada, 3Buckman, Vaudreuil-Dorion , Quebec, Canada, 4PROTEO,
Québec, Quebec, Canada
Carboxylesterases from thermophiles have become objects of special interest for structural investigation
and for a broad range of biotechnological applications. Their range of physicochemical properties makes
them enzymes of great interest for textile, food and pulp and paper industries. Carboxylesterases (EC
3.1.1.1) belong to a class of hydrolases adopting the α/β hydrolase fold. These enzymes are active at
alkaline pH and high temperature. Understanding the relationship between structure and enzymatic
properties is essential in order to produce novel biocatalysts mutants with desirable properties for a given
application. Five mutants of the recombinant Geobacillus thermodenitrificans carboxylesterase (EstGtA2)
were generated by site-directed mutagenesis in order to study the contribution of specific salt bridges in
the stabilization and refolding of EstGtA2. One particular mutant (M1a), in which a salt bridge has been
disrupted by a point mutation (R37A), was found to have an increased melting temperature. This is an
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POSTER ABSTRACTS
unexpected change in enzymatic properties considering the fact that salt bridges often contribute to
thermal stability. In order to better understand the impact of this point mutation (R37A), yield production
has been optimized for both the wild type protein EstGtA2 and the mutant M1a (R37A). Biophysical and
biochemical studies were performed to enable better understanding the structure-function relationships.
The enzymes were characterized by circular dichroism for conformational analysis, by enzymatic assays for
activity and specificity and by dynamic light scattering to determine solution conditions which are optimal
for the sample to be monodisperse. In the near future our goal is to determine the three dimensional
structures by X-ray crystallography, allowing for a detailed investigation of structural key elements which
control the properties of the carboxylesterase EstGtA2.
POST 03-258
Up-Regulation Of Rich1 Causes S-Phase Arrest And Reduces Cell Adhesion In Epithelial Cells
Lin Ming-ming, Zhang Qian-ying, Wang Yun-hong, Li Xin, Zhang Jun
Institute of Molecular Medicine and Oncology, Chongqing, China
Rich1, a previously identified Rho GTPase-activating protein (GAP) domain-containing protein, was found
to have close relationship with Rho GTPase family members in an array of cellular processes. In addition to
GAP domain, the protein also contains a serine/threonine/proline-rich domain, a SH3-binding motif and
an N-terminal BAR domain and plays a role in stimulating GTP hydrolysis to terminate Rho signaling. The
regulation functions of Rich1 in epithelial cells are still not completely clear. In the present investigation, it
was shown that up-regulation of Rich1 expression to over 3-fold by transient transfection of full-length
Rich cDNA vector caused a slight S-phase arrest with corresponding growth inhibition, but strongly
reduced cell adhesion in each of the six types of epithelial cells including HL7702, Hela, 293T, A594, H1299
and T24 enrolled in the experiments. Further in-depth survey in HL7702 cells revealed that up-regulation
of Rich1 could greatly increase the GTPase activities of both the CDC42 and RAC1, which could attenuate
the Rho protein (CDC42 and RAC1) signaling, and subsequently lowered the phosphorylation level of
ERK1 as well. A truncated Rich1 construct without GAP domain could not result in any of the biological
effects. Co-localization of Rich1 with CDC42 and RAC1 could be observed in cytoplasm respectively.
According to the discovery, it is indicated that Rich1, via its GAP domain, might act as a crucial upstream
negative regulators of the Rho GTPases-ERK1 signaling triggered by CDC42 or/and RAC1 in the control of
epithelial cell cycle, growth and adhesion. Key words: Rich1; CDC42; RAC1; S-phase arrest; cell adhesion
This work is funded by the grants from Chongqing Education Commission (KJ080301), Chongqing
Science&Technology Commission (CSTC, 2010BB5366) and National Natural Science Foundation of China
(NO. 20803098).
POST 03-259
Tracking Wood Fibers Decrystallization With Carbohydrate Binding Module
Yannick Hébert-Ouellet1, 2, Vinay Khatri1, 2, Fatma Meddeb-Mouelhi1, 3, Marc Beauregard1, 2
1
UQTR, Trois-Rivières, Quebec, Canada, 2PROTEO, Québec, Quebec, Canada, 3Buckman North America,
Vaudreuil-Dorion, Quebec, Canada
There are many steps in the complex process of converting wood into paper. One of these steps is
refining. Refining is essential for modifying the characteristics of wood fibers so that it may form paper
sheet with a specific set of properties. One important modification of wood fibers is the fibrillation of the
exposed S2 layers which promote the formation of hydrogen bonds and increase the available bonding
surface. This modification also partially converts crystalline cellulose into amorphous cellulose. On the
other hand, the majority of the energy devoted to paper manufacturing is consumed by the refining of
wood fibers. Therefore, improving energy efficiency through the understanding of wood fibers refining at
a molecular level is of paramount importance for the pulp and paper industry. In this study, we specifically
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POSTER ABSTRACTS
track the decrystallization of cellulose on the surface of refined paper sheets through the utilization
of Clostridium thermocellum CipA carbohydrate binding module 3a fused to eGFP. Correlations between
crystalline cellulose quantification, energy of refining and paper physical properties are shown.
Advantages, limitations and applications of this assay to enzymatic prerefining are also discussed.
POST 03-260
Evolution of Structure and Mechanistic Divergence in Di-Domain Methyltransferases from
Nematode Phosphocholine Biosynthesis
Soon Goo Lee, Joseph M. Jez
Biology, Washington University in St. Louis, St. Louis, Missouri, US
The phosphobase methylation pathway is the major route for supplying phosphocholine to phospholipid
biosynthesis in plants, nematodes, and Plasmodium. In this pathway, phosphoethanolamine Nmethyltransferases (PMT) catalyzes the sequential methylation of phosphoethanolamine to
phosphocholine. In the PMT, one domain (MT1) catalyzes methylation of phosphoethanolamine to
phosphomonomethylethanolamine and a second domain (MT2) completes the synthesis of
phosphocholine. The x-ray crystal structures of the di-domain PMT from the parasitic
nematode Haemonchus contortus (HcPMT1 and HcPMT2) reveal that the catalytic domains of these
proteins are structurally distinct and allow for selective methylation of phosphobase substrates using
different active site architectures. These structures also reveal changes leading to loss of function in the
vestigial domains of the nematode PMT. Divergence of function in the two nematode PMT provides two
distinct anti-parasitic inhibitor targets within the same essential metabolic pathway. The PMT from
nematodes, plants, and Plasmodium also highlight adaptable metabolic modularity in evolutionarily
diverse organisms.
POST 03-261
ERK1 and 2 - Exploring Isoform Differences
Jen Liddle, Natalie Ahn
University of Colorado Boulder , Boulder, Colorado, US
The MAP kinase pathway includes multiple isoforms of each member in the pathway for most vertebrates,
raising the question of whether these isoforms are redundant or have evolved unique functional niches.
The prototypical MAP kinases, Erk1 and Erk2, share ~ 85% sequence identity, are activated by the same
stimuli, and phosphorylate the same set of substrates. However, mouse knockout models show strong
phenotypic differences, and incomplete redundancy is shown by a strong preference for Erk2 in specific
contexts, such as T-cell proliferation, mouse embryonic survival, platelet signaling, thymocyte maturation,
and EMT capabilities. A significant body of work has established multiple methods that could provide
distinct regulatory mechanisms in vivo, including tissue-specific differential expression, nuclear
translocation rates, and the possibility of unique scaffolding interactions. However, biophysical data
suggest that intramolecular mechanistic differences can provide an additional and novel layer of signaling
regulation. We present our results characterizing the differences between Erk1 and Erk2 using hydrogenexchange mass spectrometry, mutagenesis, and kinetic assays of autoactivation as well as substrate
phosphorylation.
181
POSTER ABSTRACTS
POST 03-262
Chemical Modification of MitoNEET
Megan Laffoon1, Michael Menze2, Mary Konkle1
1
Chemistry, Eastern Illinois University, Charleston, Illinois, US, 2Biology, Eastern Illinois University,
Charleston, Illinois, US
MitoNEET, a [2Fe-2S] cluster protein, has the capability to bind to the antidiabetic drug
pioglitazone. Possible cellular functions of mitoNEET include as an electron-transport protein or an ironsulfur transfer protein. The ligation of the [2Fe-2S] cluster of mitoNEET is unique in using one histidine
and three cysteine residues. Recombinant expression of mitoNEET was accomplished using e. Coli bacteria
and was subsequently purified by using affinity fast protein liquid chromatography. After expression and
purification, UV-visible spectroscopy is used to observe the local environment of the cluster through
observation of the ligand to metal charge transfer (LMCT) bands. MitoNEET was reacted with the chemical
modifier diethylpyrocarbonate (DEPC) using a range of pH values. DEPC reacts with histidine to form a
chromophore at 250 nm. However, a change in the LMCT bands after DEPC addition indicates
modification of His87, the ligating histidine of mitoNEET. Additionally, the reactivity of the cysteine
residues of mitoNEET, all of which ligate the metal cluster, were evaluated against the biologically-relevant
molecules glutathione and fumarate. The goal of chemical modification of mitoNEET is to investigate the
reactivity of the ligating residues of mitoNEET and to understand how these modifications impact protein
stability and chemical reactivity towards small molecules like pioglitazone and macromolecules like
protein binding partners of mitoNEET.
POST 03-263
Biochemical Basis For The Extended Spectrum Cephalosporinase Activity Of A Clinical AmpC βlactamase Variant
Jozlyn R. Clasman, Brianna M. Jackman, Cynthia M. June, Rachel A. Powers, David A. Leonard
Grand Valley State University, Allendale, Michigan, US
Class C β-lactamases provide antibiotic resistance to a diverse array of microbial species through their
hydrolytic action against a wide-range of penicillin and cepholosporin β-lactam drugs. These β-lactamases
can also expand their spectrum of activity through mutations that evolve under the pressure of antibiotic
treatment. One example of this phenomenon is the appearance of a serine → asparagine mutation
(S287N) in the AmpC β-lactamase during an outbreak of E.coli in France. Subsequent analysis revealed
that the S287N variant conferred a high level of ceftazidime resistance on E.coli compared to wild-type
AmpC. Also, steady-state kinetic analysis revealed that oxyiminocephalosporins and imipenem have
lower KMvalues for the variant compared to wild-type suggesting that these drugs might bind the active
site with higher affinity in the presence of the mutation. In order to investigate the structural basis of
these interesting phenotypes, we used X-ray crystallography to determine the acyl-intermediate structures
of AmpC S287N with ceftazidime and cefotaxime bound (1.88 Å and 1.97 Å, respectively), and compared
our results to the previously-published structure of the ceftazidime acyl-intermediate of wild-type
AmpC. Our analysis indicates that the S287N mutation allows the loop formed by residues 286-296 to
adopt a new conformation, greatly relieving the steric clashes between the exocyclic carbon of the
ligand’s dihydrothiazine ring and the side-chain of residue N289. These results may lead to the design of
new cephalosporins that are less susceptible to the hydrolytic action of class C β-lactamases.
182
POSTER ABSTRACTS
POST 03-264
Common Substitutions Enhance The Carbapenemase Activity Of Oxa-51-Like Class D β-lactamases
From Acinetobacter SPP
Joshua M. Mitchell, David A. Leonard
Carbapenem resistance in Acinetobacter baumannii is a growing threat to effective treatment of
infections. Acquired carbapenemases, particularly OXA-23, OXA-24/40 and their variants are a major
source of resistance, especially when combined with porin deletion. A.baumannii has a chromosomal
carbapenemase (OXA-51), though its weak activity against most β-lactams has allowed it to escape the
high degree of scrutiny given to other carbapen-hyrolyzing class D β-lactamases (CHDLs). With more
than 50 clinical variants of OXA-51 documented, it appears that many mutations have arisen
independently several times and are sometimes associated with an increase in the minimum inhibitory
concentration values for carbapenems. Based on homology with other β-lactamases, many of these
clinical mutations are predicted to be in the active site. Starting with an OXA-51 gene, we synthesized,
expressed and purified the I129L and L169V variants of OXA-51. Steady-state kinetic analysis shows that
OXA-51 displays high KM and low kcat values for both penicillins and cephalosporins. OXA-51 affinity for
carbapenems appears to be tighter (KM 5-150 µM), although much weaker than that seen for OXA23/OXA-24/40 (doripenem KM10-30 nM). Binding and turnover of penicillins and cephalosporins were
little improved for either of the variants, but carbapemem KM values (and KS values) were much lower for
both mutants, suggesting greatly increased affinity that approaches or equals that seen with OXA-24/40
and OXA-23. The increased affinity for both mutants with respect to substrates containing α-hydroxyethyl
groups can be explained by mutation-induced remodeling of the active to better accommodate that
group.
POST 03-265
Exploring the Potential of Arylboronic Acids as Inhibitors of OXA-24 β-lactamase
Josephine P. Werner, Rachel A. Powers
Chemistry, Grand Valley State University, Allendale, Michigan, US
β-lactam antibiotics, like penicillin, are crucial to the field of medicine. Yet due to over-prescription, many
bacteria are now resistant to them. The most widespread resistance mechanism to β-lactams is expression
of β-lactamase enzymes. β-lactamases hydrolyze the amide bond of the defining four membered β-lactam
ring, rendering the antibiotic inactive. One way to overcome resistance is through inhibition of βlactamase enzymes. However, current inhibitors for β-lactamases also contain a β-lactam ring. Therefore, a
critical need exists for a novel, non-β-lactam inhibitor. The carbapenem-hydrolyzing class D β-lactamases
(CHDLs) are of particular concern, given that they efficiently hydrolyze the newer carbapenem antibiotics.
Unfortunately, these enzymes are not inhibited by clinically available β-lactamase inhibitors. Boronic acids
are known to be transition state analogue inhibitors of class A and C β-lactamases, but they have not
been extensively characterized as inhibitors of the class D β-lactamases. Boronic acids do not contain a βlactam ring in their structure, providing a novel way to inhibit class D β-lactamases. We ordered and
tested thirteen arylboronic acids for inhibition of the CHDL OXA-24. Several were identified as inhibitors
of OXA-24, with KI values ranging from 33.7 mM to 6.23 μM. These arylboronic acids serve as a starting
point in optimization efforts in the development of a novel series of inhibitors for class D β-lactamases.
183
POSTER ABSTRACTS
POST 03-266
Structure-based Discovery of a Novel Inibitor of OXA-1 β-lactamase
Leslie A. Wyman, Neil V. Klinger, Rachel A. Powers
β-lactams, like penicillin, are the most clinically prescribed antibiotics. However, due to their overuse,
resistance has developed. β-lactamase enzymes are the most common resistance mechanism used by
bacteria to combat the effects of these drugs. These enzymes efficiently hydrolyze the β-lactam ring that
defines this class of antibiotics. In response, β-lactamase inhibitors were created to disrupt this type of
bacterial resistance. Alone, β-lactamase inhibitors have minimal antibiotic activity, but when given in
combination with a partner β-lactam, they enable the antibiotic to work by inhibiting the β-lactamase
enzymes produced by resistant bacteria. Unfortunately, the structures of the inhibitors also contain a βlactam ring. The chemical similarity has allowed for resistance to develop against the inhibitors as well.
Additionally, these compounds do not inhibit members of the class D β-lactamases. Therefore, there is an
urgent need for the discovery of a novel β-lactamase inhibitor that does not resemble a β-lactam. A
structure-based approach was used to discover possibilities for potential novel β-lactamase inhibitors of
the class D β-lactamase OXA-1, a key clinical target. The program DOCK was used to screen the ZINC
database of commercially available compounds. Twenty-one compounds from the fragment subset were
ordered and tested experimentally for inhibition of OXA-1. Of the twenty-one fragments tested, seven
inhibited OXA-1 with a Ki < 5 mM. Subsequently, the structure of OXA-1 in complex with
fragment 19 (0.469 mM) was determined to 1.98 Å resolution. Using this structure as a template,
optimization of a novel series of OXA-1 inhibitor is currently underway.
POST 03-267
Kinetic And Biochemical Investigations Of Thermostable Acid Phosphatase From Zea Mays And
Glycine Max Cotyledons: A Therapeutically Important Enzyme
Nidhee Chaudhary1, Subhash Chand2, Nameet Kaur1
1
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, Uttar Pradesh,
India, 2Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT),
Delhi, New Delhi, India
Acid phosphatases (APs), a group of ubiquitous enzymes found in many plant, animals and
microorganisms, catalyze and facilitate important physiological changes within cells. Human APs are
normally found at low concentrations. However, pronounced changes in their synthesis occur in particular
diseases, where unusually high or low enzyme expression is seen as part of the pathophysiological
process. Deficiency of APs causes various diseases like; Osteomalacia, Rickets, Bone malformations. In the
present investigation few kinetic and biochemical parameters of acid phosphatase (APs) from germinating
seedlings (cotyledons) of Zea mays and Glycine max named as AP I and AP II, respectively, have been
studied. The extracted APs from the two sources were partially purified into two fractions each; based on
0-40% and 40-80% saturation level of (NH4)2SO4. The 0-40% fraction (both cases) was found to have
-1
-1
higher specific activity; the values being 88.86 units mg and 97.56 units mg in AP I and AP II,
0
0
respectively. AP I and AP II were found to be thermally stable up to 75 C and 60 C, respectively. Values of
Km, temperature coefficients (Q10) and activation energy (Ea) were found to be 1.42, 1.25 mM; 1.7, 1.5 and
-1
44.7, 40.6 kJmol for AP I and AP II, respectively. Out of the tested detergents viz.; Sodium Lauryl Sulphate
(SLS), Cween-60, Tween-80 and Tween-20; SLS acted as strong inhibitor of both; AP I and AP II, with
-4
-4
inhibition constants (Ki) 7.6 x 10 M and 2.2 x 10 M, respectively. Various metal ions (salts) inhibited AP I
2+
2+
2+
2+
+
2+
and
AP
II
activity
in
the
order
of
Mg >Cu >Zn >Ca >Na >Ba and
2+
2+
2+
2+
+
2+
2+
Cu >Zn >Mg =Mn >Na >Ba >Ca , respectively. The values of inhibition constant (Ki) for first two
2+
2+
2+
2+
-5
-5
potential inhibitors of AP I (Mg , Cu ) and AP II (Cu , Zn ) were found to be 1.7 x 10 , 2.5 x 10 M and
184
POSTER ABSTRACTS
-5
-5
3.7 x 10 , 8.5 x 10 M, respectively. These results suggest that the APs which play an important role in
energy transfer and release of inorganic phosphate are strongly affected by certain metal ions and
detergents. This study may provide direction in knowing various aspects of APs from very commonly
consumed crop plants, their potential in future development of food supplements, lead to a further
understanding of their function and may serve to extend the therapeutic application of these enzymes.
Above all, the high thermo stability enhances their importance making them industry friendly with good
economics.
POST 03-268
Investigation Of The Dynamic Amino Acid Networks in a (β/α)8 Barrel Enzyme
Jennifer M. Axe, Kathleen F. O'Rourke, Eric M. Yezdimer, Nicole E. Kerstetter, Xianrui Yuan, David D.
Boehr
Chemistry, Penn State University, University Park, Pennsylvania, US
Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent
interactions. We used nuclear magnetic resonance chemical shift correlation analyses to identify longrange amino acid networks in the alpha subunit of tryptophan synthase, both for the resting state (in the
absence of substrate and product) and for the working state (during catalytic turnover). Additionally, we
compared the networks present in the wild-type enzyme to that of the T183V variant (resulting in the
severing of an essential hydrogen bond between the β2α2 and β6α6 active-site loops). The amino acid
networks that stretch from the surface of the protein all the way into the active-site are different between
the resting andworking states and the loss of the hydrogen bond between the dynamic β2α2 and β6α6
loops results in substantial changes to the network surrounding catalytic residue Glu49. Modification of
surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and
leads to changes in catalytic rates. The amino acid networks are likely important for coordinating
structural changes necessary for enzyme function and regulation.
POST 03-269
An Uncommon Phytochelatin Synthase Gives Hints On How To Improve Their Catalytic Efficiency
On Heavy Metal Hyperaccumulator Organisms
Jorge D. Garcia1, David G. Mendoza-Cozatl2, Rafael Moreno-Sánchez1
1
Biochemistry, Instituto Nacional de Cardiologia "Ignacio Chavez", Mexico City, Distrito Federal,
Mexico, 2Plant Sciences, University of Missouri, Columbia, Missouri, US
Phytochelatin synthase (PCS) produce GSH polymers (phytochelatins, PCs) that bind and inactivate heavy
metals. Despite being a very slow enzyme, PCS is essential for cadmium detoxification and
2+
accumulation. Euglena gracilis is an aquatic protist that synthesizes PCs to hyper-accumulate Cd . We
have cloned and characterized the recombinant EgPCS, finding significant differences compared to plant
PCSases. In contrast to all previous characterized PCSases, EgPCS functions as a monomeric enzyme able
2+
2+
to synthesize PC2 to PC4, in the presence of Zn , Cd or S-methyl-glutathione (S-methyl-GS). Kinetic
analysis of EgPCS suggests a two-substrate reaction mechanism for PC2 synthesis with Km values of 18
mM for GSH and 2 mM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and
catalytic efficiency with Zn-GS2 (instead of Cd-GS2). Moreover, its heterologous expression in Cd-sensitive
2+
yeast conferred resistance to Cd . Phylogenetic analysis showed that EgPCS is distant from plants and
other photosynthetic organisms, suggesting that it evolved independently.
The EgPCS N-terminal domain contains a typical catalytic core (Cys-70, His-179 and Asp-197), suggesting
that the acyl-enzyme intermediary is formed using a GSH molecule, as reported for other PCSases. Our
results indicated that the intermediary was exclusively formed with GSH because no activity was observed
using g-EC or PC2 as substrate. An EgPCS mutant comprising only the N-terminal 235 amino acid residues
185
POSTER ABSTRACTS
(EgPCS_Nter235aa) was inactive, while the chimera AtPCS_Nter218aa::EgPCS_Cter241aa showed activity with CdGS2 but not with Zn-GS2, suggesting that the Zn-GS2 specificity is conferred by the EgPCS_Nter
domain. Additionally, this chimeric enzyme showed an optimal pH (7.5) closer to physiological
pH versus optimal pH for AtPCS (8) and EgPCS (≥8). We conclude that EgPCS showed typical features of
PCSases (conserved N-terminal domain and kinetic mechanism), but also exhibited distinct characteristics
such as preference for Zn-GS2 over Cd-GS2 as co-substrate, a monomeric structure, and ability to solely
synthesize short-chain PCs. Our results also suggested that the C-ter domain is essential for
activity/stability in a monomeric PCS and that biochemical engineering on this domain may lead us to
obtain an enzyme with higher catalytic efficiencies under physiological conditions
POST 03-270
Nonlocal Effects Of Metal Ion Binding At The Catalytic Site Of A Protein-DNA Complex.
Kaustubh Sinha1, 2, Michael R. Kurpiewski2, Sahil Sangani1, Andrew D. Kehr1, Gordon S. Rule1, Linda JenJacobson2
1
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania,
US, 2Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, US
Protein- DNA interactions play a critical role in a variety of biological processes. These interactions are
important for chromatin structure, gene regulation, DNA repair and modification and also recombination.
2+
Divalent metal ions, especially Mg , are an important cofactor in a large number of nucleic acid
processing enzymes (polymerases, nucleases, restriction enzymes, recombinases, transposases etc.). They
are not only involved in catalysis (activation of a nucleophile and stabilization of the leaving group) but
also make the formation of specific protein-DNA complexes energetically more favorable. Even though
the crystal structures of a number of nucleic-acid processing enzymes have been solved, there is a void in
our understanding of the effect of metal ion binding on the structure (and dynamics) of protein-DNA
complexes. Does metal ion binding affect only the residues close to the metal ion binding site or does it
trigger structural and dynamic changes at sites far removed from the binding site? We have used EcoRV, a
well characterized restriction endonuclease, as a model to study the effect of metal ion binding on
protein-DNA complexes. Binding studies and NMR have been used to characterize the effect of metal
binding to the EcoRV-DNA complex. We have shown that lanthanide ions competitively inhibit the
cleavage of DNA and occupy sites similar to the divalent ions. Lanthanides are thus excellent candidates
for replacing the divalent metal ions to form inactive complexes. We show that each active site can
3+
3+
2+
3+
accommodate 2 smaller Lu or Gd ions (size similar to Mg ) but only one larger La ion (size similar to
2+
Ca ). Chemical shift perturbation studies show that the metal binding induces structural changes at distal
regions of the protein. Deuterium exchange and proton exchange studies show that the metal ion binding
also causes long range dynamic changes in the complex.
POST 03-271
Recognition and Conversion of Flunitrazepam by Cyp3A4 is Altered by Caffeine
Mark Volker, Lauren Sparks, Larry R. Masterson
Chemistry, Hamline University, Cambridge, Minnesota, US
Flunitrazepam (FNTZ, also referred to as ‘roofies’) is prescribed to treat insomnia due to its strong
sedative effects. However these effects can be abused in social settings when FNTZ is administered with
predatory intent in alcoholic beverages, resulting in enhanced durations of sedation and amnesia. These
beverages often contain a combination of ethanol and the stimulant caffeine, both of which have been
shown to interact with the enzyme involved in the metabolism of FNTZ, Cyp3A4. In this study, the
steady-state kinetics of Cyp3A4 conversion of FNTZ was measured in the presence of ethanol, ethanol
186
POSTER ABSTRACTS
and caffeine, and caffeine alone. In addition, the binding thermodynamics of FNTZ with human Cyp3A4
was measured under these conditions. The rate of formation for the two products of Cyp3A4-mediated
catalysis, desmethyflunitrazepam (DM FNTZ) and 3-hydroxyflunitrazepam (3-OH FNTZ), was shown to
decrease in the presence of caffeine. Specifically, a 4-fold decrease in the rate of DM FNTZ formation
was observed (kcat changed from 1.15 min-1 to 0.27 min-1 in the presence of caffeine), while a small but
significant change in the rate of 3-OH FNTZ formation was observed (kcat changed from 3.90 min-1 to
2.87 min-1 in the presence of caffeine). Ethanol, and the combination of ethanol and caffeine, was also
shown to diminish Cyp3A4 activity. Changes in the absorbance spectrum for the heme cofactor at the
active site of Cyp3A4 were used to probe the binding affinity of FNTZ. The recognition of FNTZ by
Cyp3A4 was characterized by a Kd of 22.0 µM. In the presence of caffeine, the absorbance spectra
revealed a change in the binding mode of FNTZ to Cyp3A4 and a concomitant increase in Kd to 185
µM. The increase in Kd in the presence of caffeine indicates a decrease in the stability of the complex,
possibly due to a reorientation of FNTZ at the enzyme active site, as has been shown with the substrate
acetaminophen. Importantly, these factors indicate that product formation occurs at a diminished rate
in the presence of caffeine, providing a possible biochemical mechanism for the prolonged effects
observed during the misuse of FNTZ.
POST 03-272
Fluorogenic Probes for Mycobacterial Esterase Profiling
Katie Tallman, Kimberly Beatty
Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon, US
Fluorogenic probes go from a dark to a bright state following enzyme-mediated hydrolysis and provide a
rapid readout of enzyme activity. We have developed three novel fluorogenic esterase probes: two
derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), and
one from 2’,7’-dichlorofluorescein (DCF). Notably, DDAO excites above 600 nm but does not require
near-infrared detection capabilities. We spectrally characterized and validated our probes with a panel of
commercially available esterases and lipases. All three probes were efficiently hydrolyzed in the presence
of enzyme, but resisted spontaneous cleavage in aqueous solution. We used these probes to identify
differences in enzyme activity patterns produced by a variety of mycobacterial species, including members
of the Mycobacterium tuberculosis (M. tb.) complex. We anticipate that these probes will enable us to
annotate the M. tb. proteome under diverse conditions and to identify new diagnostic targets.
POST 03-273
Visualizing Cell Interactions With Genetically Encoded Bioluminescent Tools
Krysten A. Jones1, David Li2, Elliot E. Hui2, Mark A. Sellmyer5, Jennifer A. Prescher3, 1, 4
1
Molecular Biology and Biochemistry, Univeristy of California Irvine, Irvine, California, US, 2Biomedical
Engineering, University of California Irvine, Irvine, California, US, 3Chemistry, University of California
Irvine, Irvine, California, US, 4Pharmaceutical Sciences, University of California Irvine, Irvine, California,
US, 5Radiology, University of Pennsylvania, Philadelphia, Pennsylvania, US
Cell-cell interactions underlie diverse physiological processes and disease mechanisms, including immune
function and cancer metastases. Despite their critical importance, cell-cell contacts are difficult to
examine in live organisms without invasive procedures. Bioluminescence imaging provides adequate
sensitivity to examine small numbers of cells in vivo, but lacks sufficient spatial resolution to image
microscopic cell contacts. To re-tool this technology for visualizing cellular interactions, we engineered
bioluminescent proteins to produce light only when two distinct cell populations are nearby. These probes
comprise “split” fragments of Gaussia luciferase (Gluc) fused to the leucine zipper domains of Fos and
Jun. When cells expressing the Gluc fragments are in close proximity, Fos and Jun facilitate rapid
187
POSTER ABSTRACTS
assembly of functional Gluc, and thus light emission. The split Gluc reporters were used to repot on cellcell interactions in vitro and in models of metastatic disease. Such studies are enhancing our
understanding of cell-cell interactions relevant to human health and disease.
POST 03-274
Specific Inhibition of Enolase From Entamoeba histolytica
Normande Carrillo-Ibarra2, 1, Cesar Augusto Sandino Reyes-Lopez2, Jose Correa-Basurto1, Elibeth Mirasol
Melendez2, Claudia Guadalupe Benitez Cardoza2
1
Medicine, Instituto Politecnico Nacional, Mexico City, Mexico, 2Biochemistry, Instituto Politecnico
Nacional, Mexico City, Mexico City, Mexico
Entamoeba histolytica is the enteric protozoan that infects approximately 500 million people worldwide,
of which nearly 50 million become ill, resulting in almost 100,000 deaths annually. Clinical manifestations
include diarrhea, dysentery and in some cases, liver, brain or lung abscesses. Metronidazole is an effective
therapy, yet some parasites do persist in 40-60% of treated patients. Also, there are some reports
indicating the generation in vitro of strains resistant to Metronidazole. These reasons make new
therapeutic strategies an urgent need. Enolase is the enzyme that catalyzes the reversible dehydration of
2-phosphoglycerate to phosphoenol-pyruvate, using magnesium as a cofactor. It also participates in
different physiopathological processes, revealing enolase as a virulence factor in amoeba, since it has
been described as a plasminogen receptor, allowing the cellular invasion of the pathogen. Since E.
histolytica depends on glycolysis, enolase is an attractive therapeutic target for rationally designed drugs.
Therefore, we sought to determine structural differences between both amoeba and human enolases,
using molecular dynamics simulations. Using such differences, we are screening in silico many
compounds. Those with the best scores will be probed in vitro to find the best inhibitors.
POST 03-275
Effects of Intrauterine and Extrauterine Exposure to 1800 MHz GSM-Like Radiofrequency Radiation
on Liver Regulatory Enzymes Activities in one-month-old male New Zealand Rabbits
Nuray N. Ulusu
Koç University, Istanbul, Turkey
In recent years, people are more exposed to Radiofrequency Radiation (RFR) from raising wireless
communication technologies. Public concerns about the possible bio-effects of RFR have risen
dramatically over the past two decades and this largely depends on the widespread use of advanced RF
devices in people's everyday life. However; intense exposure to RF radiation may negatively affect the
vulnerable people of population. Firstly; children have developing and growing tissues and organs, they
are more sensitive to non-ionizing radiation impacts than adults. Children absorb more the Radio
Frequency (RF)/Microwave (MW) energy in their tissues than adults at Wi-Fi frequencies. Children’s
skulls are thinner, their brains are smaller, and their brain tissue is more conductive. Children especially
are vulnerable to RF exposure, because of the susceptibility of their developing nervous systems. Radio
Frequency (RF)/Microwave (MW) radiation may penetrate children’s heads. Secondly, children have
been using mobile phones much earlier and longer than adults. Actually, their exposure has begun as
fetus, due to the widely used telecommunication systems during the pregnancy of their mother. With
the increment in the daily life usage of telecommunication systems, concerns about the adverse effects
of RFR on children in growing age have also been increased. In this study, investigation of the possible
bio-effects of RFR on the developing male infants was aimed. A totally thirty-six New Zealand White
male rabbits, one- month old, were randomly divided into four groups which are composed of 9 rabbits.
1800 MHz GSM-like RF whole- body exposure for 15 min/day during two weeks was applied to offspring
Group II and Group IV in accordance with the experimental protocol that was reviewed and approved by
188
POSTER ABSTRACTS
the Laboratory Animal Care Committee of Gazi University. Offspring Group I and Group III were also kept
in plexiglas cage under experimental setup for 15 min/day during two weeks but device was switchedoff. The activities of enzymes related with pentose phosphate pathway; glucose-6-phosphate
dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione-dependent metabolism enzymes;
glutathione peroxidase, glutathione reductase, glutathione S-transferase, were measured in liver tissues
of young male rabbits.
POST 03-276
Imaging Protein-Protein Interactions, Post-Translational Modifications, and Non-Protein
Biomolecules by CorrelativeLight and Electron Microscopy
John T. Ngo1, Daniela Boassa2, Stephen R. Adams1, Thomas J. Deerinck2, Sakina F. Palida1, Varda LevRam1, Mark H. Ellisman2, Roger Y. Tsien1, 3
1
Department of Pharmacology, University of California, San Diego, La Jolla, California, US, 2National
Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California,
US, 3Howard Hughes Medical Institute, Chevy Chase, Maryland, US
Electron microscopy (EM) achieves the highest spatial resolution in protein localization and has long been
the main technique to image cell structures with nm resolution, but making specific molecules standout
for EM has long been challenging. Recently, powerful genetically encoded tags have been introduced that
allow specific proteins to be tracked by EM via genetic fusion, in a similar manner to how green
fluorescent protein (GFP) is used to track proteins by light microscopy (LM). Tagged proteins are revealed
by tag-mediated conversion of 3-3’-diaminobenzidine (DAB) into a localized osmiophilic polymer that is
readily distinguished under the electron microscope. Available EM tags precipitate DAB either
enzymatically through peroxidase activity or via photo-generated singlet-oxygen. While the peroxidase
tag “APEX” and photo-oxidizing protein “mini-Singlet Oxygen Generator” (“mini-SOG”) are powerful tools
for “painting” individual proteins, analogous tools for marking biochemical processes, or nonproteinaceous molecular species by EM, are lacking. As complements to the existing EM-tags, we describe
two new methods: 1) a novel split-protein complementation system based on miniSOG for visualizing
protein-protein interactions and kinase activity, and 2) “Click-EM,” a new method for imaging nucleic
acids, lipids, and glycans via bio-orthogonal ligation of photo-sensitizing dyes to functionalized metabolic
analogs. Using these methods we have visualized protein-protein interactions in the nucleus at nanometer
resolution as well as DNA replication and transcription within full context of cellular ultrastructure.
POST 03-277
Evaluating The Role Of Peroxidatic Reducing Substrates In An Unusual Catalase Activity Of
Catalase-Peroxidases
Olive J. Njuma1, Elizabeth Ndontsa2, Douglas Goodwin1
1
Chemistry and Biochemistry, Auburn University, AL, Auburn, Alabama, US, 2Chemistry and
Biochemistry, Scripps Research Institute, La Jolla, California, US
Catalase-peroxidase (KatG) is a bifunctional enzyme that decomposes H2O2 in bacteria and lower
eukaryotes. By structure, KatG is highly similar to peroxidases like cytochrome c peroxidase. However,
KatG exhibits a catalase activity that rivals that of canonical catalases, an ability no other member of its
superfamily possesses. We observed the stimulation of catalase activity by peroxidatic electron donors
(PxEDs). These results were unexpected because the prevailing paradigm for this enzyme from its
discovery is that the two activities of KatG are mutually antagonistic. The synergestic effect of the PxEDs
not only points toward a mechanism for much more efficient detoxification of H2O2, but also
substantially broadens the conditions under which such a response can be produced. To evaluate the
mechanism by which PxEDs increase KatG catalase activity, we produced variants of the enzyme
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POSTER ABSTRACTS
targeting three novel features of the KatG structure. First, the M255I, Y229F, and W107F variants
prevent formation of the KatG–unique MYW covalent adduct and eliminate the enzyme’s catalase
activity. We observed that PxEDs were unable to restore any catalase activity to these variants,
indicating that PxEDs do not act as electron transfer surrogates in place of the MYW adduct during
turnover. Second, R418A targets an arginine residue whose conformation is pH dependent, pointing
toward the MYW adduct at pH 7.0 but away from it at pH 5.0. R418A showed greatly diminished
catalase activity, but this activity could be stimulated by PxEDs to an extent similar to wild-type. The
wild-type, and R418A KatGs showed the same propensity to accumulate inactive intermediates in the
absence of an appropriate PxED, but the prevention of inactivation required a much more extensive
participation of the PxED for R418A than for wild-type. These data suggested that PxEDs prevented
accumulation of inactive intermediates produced as a result of off-pathway intramolecular electron
transfer. The W321F variant was produced to block a likely route for off-pathway transfer. This variant
showed similar catalase activity to the wild-type enzyme and a similar stimulatory effect of PxEDs,
indicating that W321 is not part of a conduit for off-pathway electron transfer events. The implications
of our results for understanding the virulence and antibiotic resistance of KatG-bearing bacteria
(e.g., Mycobacterium tuberculosis) will be discussed.
POST 03-278
Pyrrolysine-Inspired Protein Cyclization
Marianne M. Lee1, Tomasz Fekner2, Jia Lu1, Michael K. Chan1
1
The Chinese University of Hong Kong, Shatin, Hong Kong, 2The Ohio State University, Columbus, Ohio,
US
The pyrrolysine technology has been used extensively to produce recombinant proteins containing a
variety of "site-specific" modifiable unnatural amino acids for both in vitro and in vivo biochemical studies.
In this study, we demonstrated its application to the production of branched cyclic protein with a tadpolelike topology. As a proof of concept, we chose the well-studied RGD tripeptide as our subject of interest
and fused it to the C-terminus of the reporter protein, mCherry. We evaluated the cellular uptake
efficiency of the cyclized mCherry-RGD, the linear mCherry-RGD, and the wild-type mCherry without the
RGD appendage using flow cytometry, and showed that the cyclization of RGD did enhance cellular
uptake of its cargo protein. Notably, our pyrrolsyine-inspired approach for cyclized protein production
circumvents some of the common limitations encountered by current approaches, such as, the
confinement of the functionalities to the two ends of proteins, thus producing only head-to-tail cyclized
proteins, the linearization of disulfide-based cyclic proteins in the reducing environment of the cytosol,
thereby limiting its targets to those of extracellular. Our proposed approach produces cyclic unit that is
formed via an isopeptide bond, which is stable inside the reducing cytosol. Significantly, since the
pyrrolsyine analog can be incorporated at virtually any position within the protein sequence, this allows
for flexible adjustment of the size of the macrocycle and the length of the sidearms. Given the important
role of cyclic peptide in therapeutic development, our approach would have great implication in the
preparation of cyclic peptide-containing therapeutics.
POST 03-279
Functional Sectors Involved In Thermal Stability And Activity In Beta-Glucosidases
Fabio K. Tamaki2, Larissa C. Textor1, Igor Polikarpov1, Sandro R. Marana2
1
Departamento de Física e Informática, Instituto de Física, São Carlos, São Paulo, Brazil, 2Department of
Biochemistry, Institute of Chemistry, São Paulo, São Paulo, Brazil
Functional protein sectors were firstly described in serine proteinases through the identification of
physically connected positions that are statistically coupled. Each sector contained single positions
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POSTER ABSTRACTS
involved in enzyme specificity, catalysis or thermal stability. Here it is shown that functional sectors are
also present in beta-glucosidases. Indeed, eighteen statistically coupled positions in beta-glucosidase
were changed by alanine and allowed us to identify 2 functional sectors, one involved in thermal stability
(as demonstrated by Tm changes) and a second one in catalysis and in specificity modulation (determined
by kcat/Km changes associated to structural analysis). Differently of that observed for serine proteinases,
the sector involved in thermal stability does not contain physically connected positions, but is
concentrated in loops and preferentially occupied by prolines. On the other hand, the sector involved in
enzyme activity forms a layer surrounding the active site of the beta-glucosidases, so these statistically
coupled positions may modulates activity via direct contacts to the active site residues. Supported by
FAPESP and CNPq.
POST 03-280
A Chemoproteomic Platform To Quantitatively Map Targets Of Lipid-Derived Electrophiles
Chu Wang1, 2, Benjamin F. Cravatt2
1
Chemical Biology, Peking University, Beijing, China, 2Chemical Physiology, The Scripps Research
Institute, La Jolla, California, US
Cells produce electrophilic products with the potential to modify and affect the function of proteins.
Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by
endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify
the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods.
Here, we describe a competitive activity-based profiling method for quantifying the reactivity of
electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach,
we identify a select set of proteins that constitute “hot spots” for modification by various lipid-derived
electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE). We show that one of these
proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme
inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by
oxidative stress.
POST 03-281
Protein Stabilization And Prevention Of Protein Aggregation By Fungal Sucrase Of Termitomyces
Clypeatus And Application In Biotechnology And Biomedical Research
Suman Khowala, Sudeshna Chowdhury, Sanjeeta Tamang, Sangita Majumdar, Rajib Majumder
Drug Development Diagnostics & Biotechnology, CSIR-Indian Insitute of Chemical Biology, Kolkata, West
Bengal, India
The aggregation and stabilization of proteins is of critical importance in a wide variety of industrial and
biomedical situations respectively and can be exploited for efficient performances and treatments of
abnormal diseases, such as Alzheimer's and Parkinson's diseases. The sucrase of the filamentous
fungus Termitomyces clypeatus was purified and characterized as smallest enzyme in the category known
so far. In the fungus Termitomyces clypeatus sucrase was co-aggregated with cellobiase, another
glycosidase, in extra- and intra-cellular fractions and affected the catalytic efficiency, stability and
conformation of the later. Cellobiase activity decreased after disaggregation from the sucrase in intra and
extra-cellular preparations and was regained partially on in vitro addition of purified sucrase when
assayed at optimum pH and temperature. Sucrase is a well known industrial enzyme, converting sucrose
to glucose and fructose, but this observation is not known for any other sucrase. Our work focuses on
the altered organization and aggregation of purified sucrase due to changes in coaggregation other
enzyme cellobiase in the fungus for better stability and activity and on in vitro prevention of aggregation
of insulin and carbonic anhydrase up to more than 83% by the sucrase. Prevention of protein aggregation
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POSTER ABSTRACTS
by sucrase was stoichiometric and more pronounced after thermal denaturation of the enzymic activity. In
presence of sucrase large soluble aggregates of whey protein constituents were reduced in size. The
enzyme also interacted with already-aggregated insulin preparation for resolubilisation, as assayed
spectrophotometrically. This is the first evidence of an extracellular fungal sucrase from an edible fungus
with such low monomeric size and preventing protein aggregation. The study will be useful to explore
new uses of the sucrase with applications in biotechnology and biomedical applications.
POST 03-282
Using Multiwavelength Collisional Quenching to Investigate the Effects of Arginine and Inhibitors
on inducible Nitric Oxide Synthase
Rachel Jones, Ellis Bell
Nitric Oxide Synthase (NOS) plays a critical role in a variety of signaling pathways and responses to
cellular challenge. Although the enzyme is well studied, the role of conformational flexibility in inducible
Nitric Oxide Synthase (iNOS) activity has received little attention. To understand the role of
conformational flexibility; experiments using multi-wavelength collisional quenching with excitation at
280nm (Tyrosine and Tryptophan) and 295nm (Tryptophan only) were performed with differently charged
quenchers (iodide, acrylamide, and cesium) were conducted in the presence and absence of the substrate
Arginine asc well as a series of know inhibitors of the enzyme, Aminoguanidine hydrochloride, 1400W
dihydrochloride and BYK 191023 dihydrochloride, to determine their effects on tyrosine/tryptophan
exposure. The collisional quenching data also suggests that Arginine increases the exposure of
tyrosine/tryptophans(non-polar regions) of the protein, consistent with Arginine increasing the flexibility
of the protein. A comparison of the effects of a number of inhibitors of iNOS on the multi-wavelength
collisional quenching suggest different structural basis for their inhibitory effects. Analysis of the amino
acid sequence of iNOS using a variety of bioinformatics approaches suggests that these changes in
flexibility could be related to the regulation of disordered regions of the protein.
POST 03-283
Enzyme Active Sites May Extend Further Than We Thought
Lisa Ngu, Penny J. Beuning, Mary Jo Ondrechen
Chemistry & ChemBiol, Northeastern University, Boston, Massachusetts, US
Understanding how nature designs enzymes to catalyze reactions under mild conditions is an important
and intriguing problem. To date the catalytic mechanisms of hundreds of enzymes have been
investigated. However it is not always obvious, even with a crystal structure with a bound substrate mimic,
which residues contribute significantly to catalysis. We have developed computational methods Partial
Order Optimum Likelihood (POOL), a machine learning methodology, and Theoretical Microscopic
Anomalous Titration Curve Shapes (THEMATICS) to predict the residues that participate in catalysis. These
methods require only the 3D structure of the query protein as input and are based on computed
electrostatic and chemical properties. Dynamic conformational changes during catalysis, in addition to
electrostatic interactions, allow for coupling between remote residues and the canonical active site
residues of an enzyme. This suggests that at least some enzyme active sites are spatially extended, with
participation by remote residues in catalysis. Guided by computational predictions and using site-directed
mutagenesis and kinetics experiments, we have shown that distal residues play significant roles in the
catalytic activity of Ps. putida nitrile hydratase, human phosphoglucose isomerase, and the E. coli Y family
DNA polymerase DinB. Here we present new computational predictions and kinetics data for E.
coli ornithine transcarbamylase (OTC). OTC is reported to undergo induced-fit conformational changes
upon binding carbamoyl phosphate, followed by binding of ornithine. POOL predicts OTC to have an
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POSTER ABSTRACTS
extended triple-layer active site. Kinetics assays of OTC variants Glu299Gln and Arg57Ala show these
POOL-predicted remote residues, located in the second and third layer respectively, are important for
catalysis. The use of POOL in the prediction of spatially extended active sites is presented.
POST 03-284
Nucleotide Pyrophosphatase/Phosphonuclease Possesses The Zeatin Cis-Trans Isomerase Activity
In Vitro
Tomáš Hluska1, Michaela Baková1, René Lenobel2, Marek Šebela2, Petr Galuszka1
1
Department of Molecular Biology, Centre of the Region Haná, Olomouc, Czech Republic, 2Department
of Protein Biochemistry and Proteomics, Centre of the Region Haná, Olomouc, Czech Republic
Metabolism of isoprenoid cytokinins has been extensively studied in the past years. However, biosynthesis
of cis-zeatin remains unknown. Besides isopentenylation of adenine in tRNA there is another option - cistrans-isomeration. In 1993, a zeatincis-trans isomerase was described, purified to near homogeneity and
partly characterised. However, the protein nor gene sequences were not identified yet. 20 years later, we
have purified the enzyme using several chromatographic columns and gel chromatofocusing. The protein
was identified as putative nucleotide pyrophosphatase/phosphonuclease. After heterologous expression
we have confirmed FAD hydrolase (nucleotide pyrophosphatase) and zeatin cis-trans isomerase activities
of the enzyme in vitro. Nevertheless its contribution to cytokinin metabolism in planta remains to be
elucidated.
POST 03-285
Exploring the Trigger for Cooperativity at the Subunit Interface of Malate Dehydrogenase
Jacqunae Mays, Ellis Bell
Malate Dehydrogenase is a homodimeric enzyme which shows negative cooperativity in terms of binding
its allosteric regulator Citrate, and is presumed to operate via reciprocating subunit mechanism,
dependent upon subunit interactions. To explore the triggers of these subunit interactions we have
constructed a number of both subunit interface, and active site mutations and are exploring ligand
induced conformational changes using a variety of probes of conformation and conformational flexibility
including ANS binding and fluorescence, limited proteolysis, and using positional tryptophan mutants (the
native protein lacks tryptophan), multiwavelength collisional quenching techniques with excitation at
either 280nm (excitation of tyrosine and tryptophan and a sensitive probe of tyrosine to tryptophan
resonance energy transfer) or 295nm (excitation of tryptophan only). Correlation of the data on ligand
induced conformational changes with previous kinetic data for various mutants suggests that key residues
at the interface relay information from one active site, triggered by interaction of carboxylic acid
containing ligands with one of the active site arginine residues, to the adjacent active site and are
responsible for the subunit interactions involved in both catalytic activity and regulation of this enzyme.
This work was supported by NSF Grant MCB 0448905 to EB
POST 03-286
Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity
Yalemi Morales, Damon Nitzel, Owen Price, Shanying Gui, Joan Hevel
Utah State University, Logan, Utah, US
Elevated levels of asymmetric dimethylarginine (ADMA) correlate with increased markers of reactive
oxygen species in subjects with risk factors for cardiovascular disease. ADMA is generated by the
catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs),
and is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Reports have shown that DDAH
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POSTER ABSTRACTS
activity is down regulated and PRMT1 expression is upregulated under oxidative stress conditions, leading
many to conclude that ADMA accumulation occurs via increased synthesis and decreased degradation.
Here we report that recombinant PRMT1, the major PRMT isoform in humans, is susceptible to oxidation.
Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event
involves one or more cysteine residues. We demonstrate a hydrogen peroxide concentration-dependent
inhibition of PRMT1 activity which is readily reversibly under physiological H2O2 concentrations. We also
show that increased PRMT1 activity after reduction correlates with a shift towards a smaller oligomeric
state and that one or more cysteine residues are responsible for the effects of oxidation on PRMT1. Our
results challenge the unilateral view that increased PRMT1 expression results in increased ADMA
synthesis, but rather demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.
This also raises the possibility that PRMT1 could be a key player in the cellular oxidative stress response.
POST 03-287
Structural and Biochemical Characterization of thielavia Terestriscutinase (Ttc)
Abhijit N. Shirke1, 2, Danielle A. Basore3, 2, Evan Baugh4, An Su1, 2, Glen Butterfoss5, George I. Makhatadze3,
1, 2
, Christopher Bystroff3, 6, 2, Richard A. Gross1, 2
1
Chemistry & Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, US, 2Center for
Biotechnology & Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, New York,
US, 3Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, US, 4Biology, New York
University, New York, New York, US, 5Center for Genomics and Systems Biology, New York University,
Abu Dhabi, Abu Dhabi, United Arab Emirates,6Computer Science, Rensselaer Polytechnic Institute, New
York, New York, US
Cutinase, an esterase found in phytopathogenic organisms, shows potential as an industrial catalyst owing
to its ability to hydrolyze esters in rigid chains such as poly(ethyleneterephthalate) and hindered esters in
small molecules. Cutinase produced by the thermophilic fungi Thielavia terestris (TtC) displays a distinct
characteristic of being stable and active in acidic conditions as compared to other cutinases
from Fusarium Solani (FsC), Aspergillus oryzae (AoC) and Humicola insolens (HiC) which are stable and
active in alkaline conditions. In order to rationalize TtC's distinct behavior, a homology model was
developed and a thorough structural and biochemical characterization was performed based on
computational and experimental analyses. The difference stability at acidic pH was rationalized based on
surface charge calculations. With respect to enzyme activity, the optimum pH was found to be acidic and
basic for polycaprolactone hydrolysis and cellulose acetate deacetylation, respectively. This difference in
pH optima is believed to be due to differences in substrate binding. Also, the presence of glycosylation
sites and experimental detection of protein glycosylation provides an opportunity to explore the extent
that glycosylation influences the stability and activity of TtC.
POST 03-288
Investigating Molecular Determinants that Modulate the Preference of IkBs for Specific NFkBs
James D. Marion, Elizabeth A. Komives
Biochemistry, UCSD, San Diego, California, US
Cellular responses to internal and external stimuli are the result of cell signaling pathways that mediate
events critical for cell survival. Essential to these cascades are transcription factors, such as NFkB, that
regulate cellular activity and gene expression. In mammals, the NFkB family consists of different homoand heterodimers of RelA (p65), p50, p52, c-Rel and RelB, which interact with different inhibitors of NFkBs
(called IkBs) that render them transcriptionally inactive. Phosphorylation-induced ubiquitination and
subsequent degradation of IkBs liberates NFkB dimers that then translocate to the nucleus, bind DNA and
participate in transcriptional activation of numerous target genes. While a great deal of research has
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POSTER ABSTRACTS
focused on the NFkB family, the way in which cells determine which distinct NFkB dimers are present in
the cytoplasm, and are then activated to differentially induce specific target genes, has yet to be
elucidated. Collaborative research efforts amongst the Komives, Ghosh and Hoffmann labs have recently
collected data suggesting that specific IkB proteins bind preferentially to certain NFkB homo- and
heterodimers. These preliminary results led to our development of the “chaperone hypothesis” which
proposes that IkBs serve as chaperones, maintaining certain NFkB dimer populations in the cytoplasm. My
initial investigations using in vitro affinity purification assays combined with quantitative SDS-PAGE gel
analysis have revealed that subunit exchange, between NFkB homo- and heterodimers, occurs readily
when an IkB protein is not bound. While this suggests that the IkB proteins are critical for determining the
latent pool of NFkB isoforms available for activation, further investigations using NFkB constructs we have
developed for fluorescence and fluorescence anisotropy measurements, will quantify the dissociation
rates and rates of exchange between specific NFkB dimers with and without IkB proteins present. With
these data, combined with future in vivo studies, we hope to provide a greater understanding of the
underlying molecular mechanisms that modulate NFkB signaling.
POST 03-289
The Examination Of The MALDI-TOF-MS Analysis Of The Proteins And The Peptides By Use Of The
Sinapic Acid Derivatives As The New Matrix.
Narumi Hirosawa1, Takeshi Sakamoto2, Yasushi Uemura3, Yasushi Sakamoto1
1
Biomedical Research Center, Saitama Medical University, Saitama, Japan, 2Faculty of Pharmaceutical
Scieneces, Josai University, sakado, Japan, 3Exploratory Oncology Research & Clinical Trial Center,
Kashiwa, Chiba, Japan
To examine the increase in efficiency of the ionization of the proteins and peptides in matrix-assisted laser
desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis, we synthesized 15 kinds
of sinapic acid derivatives as the candidacy of the matrix and analyzed about the ability as the new
MALDI-TOF-MS analytical grade matrix of these derivatives. Some in these derivatives had unique
characteristics respectively comparing with general matrices a -Cyano-4-hydroxycinnamic acid (CHCA),
2,5-Dihydroxybenzoic acid (DHBA) and Sinapic acid(SA). A synthesized derivative (A) 2-Cyano-3-(4hydroxy-3,5-dimethylphenyl)prop-2-enoic acid was possible for ionizing of proteins. Especially, it seemed
to be effective by the ionization of large proteins but the ionization of peptide was supposed to be
weaker than SA. On the other hand, we found that (B)(2E)-3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2enoicacid, (C)(2E)-3-(2-bromo-4-hydoroxy-5-methoxyphenyl)prop-2-enoic acid and (D)(2E)-3-(2-iodo-4hydoroxy-5-methoxyphenyl)prop-2-enoic showed the sensitivity which is stronger than CHCA on proline
containing peptide. However, the ability to ionize proteins was weak. Additionally, we performed
Quadrupole ion trap MS-MS analysis of the peptides of which the derivative of these (B), (C) and (D) was
the matrix. And, these derivatives showed stronger immonium ion intensity than DHBA. Particularly the
iodination derivative (D) had the strongest ability about the occurrence of immonium ion. It seems to be
extremely useful to suppose the amino-acid sequence of peptides.
POST 03-290
Sequential Phosphorylation of SIKE by TBK1
Hyejin Park, SoHo Kim, Jessica Bell, Ellis Bell
Our recent work (Marion et al. J. Biol. Chem. (2013) 288:18612) has demonstrated that SIKE can be
multiply phosphorylated by the protein kinase, TBK1, and that phosphomimetic mutants representing
differently phosphorylated SIKE forms inhibit TBK1 utilization of its other substrate, IRF3, with significantly
different inhibition constants. To establish whether there is a sequence to the phosphorylation of SIKE by
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POSTER ABSTRACTS
TBK1, we are investigating the time dependence of site-specific SIKE phosphorylation with WT SIKE and
several phosphoknockout SIKE mutants. In time-dependent phosphorylation experiments, SIKE with
different levels of total phosphorylation was isolated by ion exchange chromatography and unique
phosphorylation sites of the separated isoforms identified by fragmentation and MALDI-TOF or tandem
mass spectrometry as appropriate. To determine if SIKE phosphorylation proceeds in a site specific
manner, six single site phosphoknockout mutants have been constructed and tested for their ability to act
as substrates of TBK1. These studies will establish whether certain phosphorylation sites play a dominant
role in subsequent multiple phosphorylation. This work is supported by VCU CCTR Endowment Funds,
R21AI107447
POST 03-291
Effects of Phosphorylation on the Structure & Stability of SIKE
Clara Kerckhove, Jessica Bell, Ellis Bell
Our recent studies (Marion et al. J. Biol. Chem. (2013) 288:18612) have indicated that SIKE is a substrate
for the protein kinase, TBK1, and can be phosphorylated at multiple sites. No 3-dimensional structures of
SIKE or related proteins are available. To investigate the impact of phosphorylation on the structure and
stability of SIKE we have used a combination of circular dichroism spectroscopy and multiwavelength
collisional quenching to investigate the conformation and flexibility of SIKE and the effects of
phosphorylation. Three different length constructs of SIKE (SIKE72-207, SIKE1-112, SIKE 113-207) and
several phosphomimetic or phosphoknockout (S6E, S6A, S4A, S2A, S185A) mutants have been constructed
and used under native conditions to study the impact of phosphorylation. Effects on stability on native
and mutant forms has been investigated by thermal melts monitored at an ellipticity of 222nm, and by
guanidine or urea unfolding, followed by fluorescence. This work is supported by VCU CCTR Endowment
Funds, R21AI107447
POST 03-292
Multiwavelength Collisional Quenching to Study Ligand Protein Interactions in Glutamate
Dehydrogenase
Chun Li, Ellis Bell
Collisional quenching approaches using neutral or charged quencher molecules have long been used to
indicate conformational changes in proteins Here we introduce a novel approach to collisional quenching
using multiwavelength analysis of spectral data collected at two different excitation wavelengths with a
variety of small molecule and protein test fluorophores. The results show that not only can detailed
information about the local environment of tryptophans in proteins be obtained (even proteins with
multiple tryptophans), but information is derived resulting from tyrosine-tryptophan resonance energy
transfer in proteins. Using Acrylamide, Iodide and Cesium collisional quenchers with both tyrosine and
tryptophan derivatives and with model proteins with one or more tryptophans we demonstrate the utility
of the approach for defining the local environment and changes induced by ligand binding, Furthermore,
during the course of these studies we revealed that pi-cation effects disrupt resonance energy transfer
between tryptophan (acting as either donor or acceptor) and other moieties in the protein. With
Glutamate Dehydrogenase we have used this technique to investigate substrate, cofactor and regulatory
ligand interactions and found that ligands which support or promote activity lead to increased flexibility
of the protein while ligands that inhibit the reaction have the opposite effect. This work was supported
by NSF Grant MCB 0448905 to EB
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POSTER ABSTRACTS
POST 03-293
Mapping The Binding Sites Of Class D beta-Lactamases For Inhibitor Design And Discovery
Joshua M. Mitchell, Rachel Powers
Beta-lactams are the most widely prescribed class of antibiotics in clinical use today, but chronic use and
misuse has led to the development of widespread resistance to these chemotherapies. Beta-lactam
resistance is conferred through the production of beta-lactamase enzymes, which hydrolyze the lactam
ring of these drugs, rendering them inactive. To overcome this resistance, beta-lactamase inhibitors have
been developed, but these inhibitors also contain a beta-lactam ring. Due to the chemical similarity that
exists between b-lactam antibiotics and b-lactamase inhibitors, the discovery and design of novel, non-blactam inhibitors offers a sensible approach to overcome this resistance. Of special concern are the class D
beta-lactamases, which are not inhibited by classic beta-lactam-based inhibitors. To address this issue, a
structure-based consensus overlay approach was employed to identify and characterize the binding sites
of OXA-1 and OXA-24/40. These maps were constructed based on the X-ray crystal structures of twentyone complexes of these enzymes with different ligands. Several binding site hot spots were identified and
used to guide the program DOCK to screen the fragment subset of the ZINC database. Twenty-one
compounds were selected and tested from the docking hit list. Four of these show inhibition of the OXA
enzymes in the low millimolar range. With further improvement and development, we are poised to
develop these compounds into the first clinical inhibitors of class D β-lactamases.
POST 03-294
Determining the Effects of MitoNEET on Cellular Dehydrogenase Activity
Sarah Banister1, Matthew Woodruff1, Paige Birge1, Michael Menze2, Mary Konkle1
1
Chemistry, Eastern Illinois University, Charleston, Illinois, US, 2Biology, Eastern Illinois University,
Charleston, Illinois, US
MitoNEET is a recently discovered mitochondrial [2Fe-2S] protein that is a binding partner of the antidiabetic drug pioglitazonme. MitoNEET contains a unique three cysteines and one histidine ligation of the
metal cluster. However, the cellular function of mitoNEET is currently unknown. Several functions have
been proposed including a role in cellular respiration, as an iron-sulfur cluster transfer protein, and as an
electron-transport protein. Since each of these functions requires a binding partner, pull-down assays
using histidine-tagged mitoNEET and cellular lysates from either mouse liver and HepG2 cells were done
to capture and identify protein binding partners of mitoNEET. Proteomic analysis identified the cellular
dehydrogenase enzymes of malate dehydrogenase 1 (MDH1) and glutamate dehydrogenase 1 (GDH1) as
putative binding partners of mitoNEET. Kinetic analysis of GDH1 and MDH1 in the presence and absence
of mitoNEET and pioglitazone was accomplished using spectrophotometric methods.
POST 03-295
In Silico And Kinetic Studies To Verify The Potency Of α-Glucosidase Inhibitors Isolated From
Morus Alba L.
Shakeel Ahmad1, Akash Chaudhary1, Shadab Ahmad2, Mohd. Tashfeen Ashraf1
1
School of Biotechnology, Gautam Buddha University, Greater Noida, India, 2School of Life Sciences,
Jawaharlal Nehru University, New Delhi, Delhi, India
Alpha-glucosidase is a carbohydrate-hydrolase that acts upon α-1,4-glycosidic bonds. It breaks down
starch and disaccharides to glucose by hydrolyzing the terminal non-reducing 1-4 linked alpha- glucose
residues. Our study aims to verify, throughIn silico and kinetic studies, the efficacy of inhibition of αglucosidase by certain phenolic compounds isolated from the polyphenol enhanced extract of mulberry
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POSTER ABSTRACTS
fruit (Morus alba L.). Our In silico studies confirmed that out of 25 compounds isolated from mulberry
fruit, as reported by Yihai Wang et. al, three of them showed significant enzyme binding, and thereby
found to be promising for kinetic studies in order to develop effective inhibitors of α*
glucosidase. Correspondence: [email protected]
POST 03-296
Molecular Dynamic Studies of the Reductase Domain of Polyketide Synthase from the
Myxobacterium Stigmatella Aurantiaca
Andrew J. Schaub1, 5, Jesus Barajas2, Ray Luo2, 3, 4, Shiou-Chaun (Sheryl) Tsai2, 5, 6
1
Biological Sciences (Gateway program in Mathematical, Computational and Systems Biology), University
of California, Irvine, Irvine, California, US, 2Molecular Biology & Biochemistry, University of California,
Irvine, Irvine, California, US, 3Biomedical Engineering, University of California, Irvine, Irvine, California,
US, 4Computer Science, University of California, Irvine, Irvine, California, US, 5Chemistry, University of
California, Irvine, Irvine, California, US, 6Pharmaceutical Sciences, University of California, Irvine, Irvine,
California, US
Polyketides are a large and diverse family of secondary metabolites, and an important source of natural
products that include antibiotics, chemotherapeutics, immunosuppressants and toxins. Polyketides are
synthesized from various starter units by polyketide synthases (PKSs). Type I modular PKSs are large
megasynthase complexes that follow biochemical instructions, which include polyketide start unit
selection, chain length control, cyclizations and postprocessing. Type I modular PKSs contain several
catalytic domains, each with its own defined function capable of being reused in multiple catalytic
cycles. Myxalamids are a class of potent electron transport chain inhibitors, which are produced by
myxobacteria (slime bacteria) using PKSs. Myxalamid S is synthesized by a type I modular polyketide
synthase and non-ribosomal peptide synthase (PKS-NRPS) system in S. aurantiaca.
Molecular dynamic (MD) studies were performed on the reductase (RE) domain of the PKS. The RE
domain is responsible for cleaving a thioester bond, and releasing the polyketide product utilizing the
cofactor NADPH as a reducing agent. The RE domain of PKS in S. aurantiaca is hypothesized to perform
[2 + 2]e reductions. MD was used to model the reductions, as well as conformational changes in the
binding pocket of TE leading to the release of the final polyketide product Myxalamid S.
POST 03-297
Mechanisms Regulating Ribosome Biogenesis by AKT and c-MYC
Simone Woods, Colin House, Gretchen Poortinga, Ross Hannan, Katherine Hannan
Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
Ribosome biogenesis is an energy consuming process essential for cell growth and proliferation, thereby
requiring tight regulation. Recently it has become evident that deregulated ribosome biogenesis can
underlie cancer susceptibility, and be specifically targeted in cancer cells [1]. The transcription factor cMYC is a master regulator of ribosome biogenesis. We have reported that c-MYC can selectively
transcribe a RNA Polymerase I (Pol I) specific regulon [2]. We recently demonstrated that the kinase AKT
also mediates Pol I-driven rDNA transcription to a similar extent as c-MYC [3]. Importantly, maximal
activation of rDNA transcription and thus cell growth was achieved through the cooperative activities of
both c-MYC and AKT [3]. The mechanism by which AKT alone and in cooperation with c-MYC regulates
Pol I transcription remains unknown. We hypothesise that AKT may phosphorylate and subsequently
activate c-MYC-regulated Pol I regulon components. We have identified nine out of sixty-six of these
components and associated factors as putative AKT substrates based on a high stringency bioinformatics
analysis using Motif scan. To test this hypothesis, we immunopreciptated the Pol I complex and
associated proteins and used high sensitivity LC-MS/MS to characterise the members of the complex
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POSTER ABSTRACTS
and to identify phosphorylated peptides. We find that endogenous nucleolar protein treacle is present
in the Pol I complex and phosphorylated at one of the putative AKT phosphorylation sites identified by
Motif Scan. Furthermore, immunoprecipitated treacle can be phosphorylated by purified AKT in vitro
demonstrating that treacle is a direct AKT substrate. Consistent with our hypothesis, the treacle gene
(TCOF1) is also a c-MYC target [2]. We are currently further validating treacle as a target of c-MYC and
AKT and assessing the contribution of increased treacle expression and AKTdependent phosphorylation
to the cooperation of AKT and c-MYC in driving Pol I transcription and ribosome biogenesis.
POST 03-298
Highly Selective Peptide Substrates For The Assay Of Cysteine Peptidases From The C1 Family
Elena N. Elpidina1, Irina Y. Filippova2, Tatiana A. Semashko1, Elena A. Vorotnikova1, Valeriya F.
Sharikova2, Konstantin S. Vinokurov3, Lyndsey Fallis4, Brenda Oppert4
1
Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russian
Federation, 2Chemical Department, Moscow State University, Moscow, Russian
Federation, 3Entomological Institute, Biology Centre AV ĈR, , Ĉeské Budĕjovice, South Bohemian Region,
Czech Republic, 4USDA Agricultural Research Service, Center for Grain and Animal Health Research,
Manhattan, Kansas, US
Cysteine peptidases are important in many biological processes. In this study, we describe the design,
synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The
structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-A, where Glp –
pyroglutamyl, A = pNA (p-nitroanilide), AMC (4-amino-7-methylcoumaride) or AFC (4-amino-7trifluoromethyl-coumaride); Xaa = Phe, Val. The synthesis of substrates was enzymatic to guarantee the
selectivity of the reaction and provided optical purity of the target compounds, thus simplifying the
scheme of synthesis and isolation of products. Hydrolysis of substrates was studied by representative
cysteine peptidases from the C1 family: plant enzymes papain, bromelain, ficin, and bovine and human
lysosomal cathepsins B and L, respectively. The synthesized substrates were selective for cysteine
peptidases of C1 family and were not hydrolyzed by peptidases of other classes: serine peptidases trypsin,
α-chymotrypsin, subtilisin Carlsberg, aspartic peptidase pepsin and metallopeptidase thermolysin. The
substrates were successfully used to monitor cysteine peptidases activity during the chromatographic
separation of a multi-component set of digestive peptidases of a beetle, Tenebrio molitor. The role of the
proposed selective substrates for the enzyme characterization in multicomponent mixtures were
demonstrated also by inhibitory studies of cysteine peptidases in the midgut extracts from T.
molitor larvae, as well as larvae of insect pests of the genus Tribolium. The results of the analysis show
that the proposed substrates are superior to other commercially available cysteine peptidase substrates in
the evaluation of biologically-active enzymes in complex systems. This work was supported by ISTC grant
3455 and RFBR grants 12-04-01562-а, 12-03-01057-a.
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POSTER ABSTRACTS
POST 03-299
Collagen Determination and Its Application for the Authentication of Tortoise Shell
Linqiu Li1, Hon-Yeung Cheung2
1
Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China, 2Department of
Biomedical Sciences, City University of Hong Kong, Hong Kong, China
Tortoise shell has been using as medicine for a thousand of years in China for its benefit of nourishing
the yin and suppressing the sthenic yang. Modern research reveals that it enhances kidney functions,
invigorates the bone and regulates menstruation. Nowadays, it is commonly utilized as an ingredient in
various kinds of functional and tonic food, e.g. tortoise jelly (Gui-ling Gao) and Gui Lu Erxian Jiao.
However, some fake tonic food is made from gelatination-food or food gums instead of tortoise shell.
Even though this tonic food is made from tortoise shell, the proper species is controversial. Therefore, it is
necessary to develop a method for authentication of tortoise shell. Since high content of hydroxyproline
(hypro) is commonly found in tortoise shell, we speculate that tortoise shell is in abundant in collagen.
Therefore, we intend to differentiate this Chinese medicine by determining and charactering the collagen.
In this work, a method for extraction and characterization of collagen in tortoise shell was developed.
After a series of treatment (removal of lipid, calcium, and non-collagen), the collagen in tortoise shell was
extracted by pepsin (in 0.5 M acetic acid, 1000U/mg) and was purified by 4% NaCl (w/v). Subsequently,
the pure collagen was determined and characterized by SDS-PAGE gel and capillary electrophoresis (CE).
As a comparison, two collagen-containing materials, chicken skin and chicken tendon, were studied at the
same time. It was found that the band pattern of SDS-PAGE gel of Cuora trifasciata (CT), Chinemys
reevesii (CR), Curoa flavomarginata (CF), and Trachemys scripta (TS) were almost the same. While one
more band appeared between 25 - 37 kD in Cuora Aurocapitata (CA) and Hardella Thurjii (HT), which were
not found in other species. Nevertheless, only a few bands (around 100 kD) were seen in chicken skin and
chicken tendon. The result of CE manifested that the content of collagen varied from 116.986 in CT to
161.333 mg/g in CF, with a range of extraction efficiency of 63.031% ~ 73.330%. By comparing the ratios
of hypro/gly, hypro/pro, hypro/hylys and gly/pro, it was concluded that the ratios of these amino acids in
collagen extracted from chicken skin and chicken tendon were distinctly different from that of tortoise
shell, which suggested that the established method could be applied for the authentication of tortoise
shell. * Content of amino acid was determined by capillary electrophoresis; 1 Standard deviation (SD) was
calculated by analyzing at least six of each material with triple replicates (Only adults were studied); 2
Hypro in extracted collagen from tortoise shell; 3 Hypro in tortoise shell raw material; extraction
efficiency= (yield of extracted collagen/total collagen) ×100%. c(collagen)=c(Hypro)/13%
POST 03-300
The B-Domain-Half-Deleted Recombinant Coagulation Factor VIII (FVIII) Shows Much Higher
Coagulation Activity Compared With B-Domain-Deleted Recombinant Ones
Zhang Jun, Zhu Chong-yang, Wen Quan, Lin Ming-ming
Department of cell biology & genetics, Chongqing Medical University, Chongqing, China
Human coagulation factor VIII (FVIII) consisting of 2332 amino acid residues is a cofactor for activation of
FX in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder
hemophilia A. FVIII contains a domain structure of A1-A2-B-A3-C1-C2. It is previously demonstrated that
most of B domain can be deleted and the resulting recombinant protein has essentially normal survival in
circulation and corrects the bleeding tendency in hemophilia A patients. Replacement therapy using Bdomain-deleted FVIII is the leading scheme in the management of hemophilia A. However approximately
15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein during
long-term treatment. Therefore it is a continuous pursuit of lower immunogenicity, higher efficacy and
200
POSTER ABSTRACTS
longer activity of B-domain-deleted recombinant FVIII in recent decades. But so far much importance is
attached to the chemical modification (including with polyethylene glycol (PEG), disulfide bond and
excipient) and shorter recombinant FVIII. In this investigation, a longer recombinant FVIII with B-domainhalf-deleted (residues 979 through 1458, designated BDHD-FVIII) was constructed, which was in turn
transfected and expressed in Chinese hamster ovary cells and purified according to the standard protocol.
The activity of BDHD-FVIII was compared with B-domain-deleted FVIII (residues 751 through 1642,
designated BDD-FVIII) and two commercial FVIII products (C-FVIII). The activity of FVIII (FVIII: C) was
determined using one-stage clotting assay. Although it was inferred that B-domain didn’t play a crucial
role in FVIII functions, the experimental results clearly indicated that HBDD-FVIII showed much higher
coagulation activity than BDD-VIII and C-FVIII. Addition of purified von Willebrand factor (vWF) did not
significantly alter the coagulation activity for all the tested samples. Highly glycosylation in specific
regions of B-domain probably involved in the activation processing and stability of FVIII might be
responsible for the results. The investigation implies that moderate length of recombinant FVIII with
higher activity could reduce the antibody reactions by minimizing the treatment dosage. From a fresh
perspective, the primary evidences promise further search for the optimal regions and lengths in Bdomain to be deleted. Key word: Coagulation factor VIII; Recombinant FVIII; activity of FVIII; vWF
POST 03-301
Novel Heterotetrameric Enzymes in Cholesterol Metabolism from Mycobacterium tuberculosis
Meng Yang1, Kip Guja2, Miguel Garcia-Diaz2, Suzanne Thomas1, Nicole Sampson1
1
Chemistry, Stony Brook University, East Setauket, New York, US, 2Pharmacological Sciences , Stony
Brook University , Stony Brook, New York, US
The genome of Mycobacterium tuberculosis (Mtb) contains a disproportionately large number of lipid
metabolizing genes. The ability of this pathogen to metabolize complex lipids like cholesterol, and the
roles that this steroid plays in the virulence and pathogenesis of this pathogen are increasingly
evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an
architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are
α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent acad (fadE) genes
that are regulated by cholesterol. We demonstrate that FadE26-FadE27 catalyzes the dehydrogenation of
an analog of the 5-carbon side chain cholesterol degradation intermediate. Sequence alignment with
ACAD homologs and the crystal structure of FadE26-FadE27 show only two FAD cofactors per tetramer
and a single active site per dimer, as previously predicted. This is the first structural report of a
heterotetrameric acyl-CoA dehydrogenase. The Mtb igr operon plays an essential role during the latent
stage of Mtb infection. Infection of macrophages or mice with an igr deletion mutant (Δigr) results in
attenuated growth early in infection. In vitro, the igr operon is required for growth on cholesterol, but not
fatty acids, as sole carbon source. Here we report the first structures of a heterotetrameric MaoC-like
enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the igr operon. We
demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, a 3-carbon side chain
cholesterol degradation intermediate, in the modified β-oxidation pathway for cholesterol side chain
N
degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2 reveal an unusual, modified
hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to
accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that
accommodate the four rings of the steroid substrate and offer an explanation for why the unusual
heterotetrameric assembly is utilized for hydration of this steroid. These unique a2b2 heterotetrameric
architectures, absent in humans, provide an opportunity to develop new anti-mycobacterial drugs that
target an important steroid metabolic pathway highly specific to Mtb.
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POSTER ABSTRACTS
POST 03-302
Energetic Contribution of n→π* Interactions to PII Conformations of Model Peptides
Liu He, Zhengshuang Shi
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan,
Hubei, China
Recent lines of evidence indicate that the backbone conformation of unfolded peptides is predisposed to
the polyproline II (PII) conformation, a structure with backbone dihedral angles (φ,ψ) centered at (-75°,
[1]
+145°) . What interactions are responsible for the predominance of PII in unfolded peptides remains a
poorly answered question. Recent lines of evidence from statistical surveys and theoretical calculations
*
suggest that the n-π interaction, together with other noncovalent interactions, might help to define and
[2,3]
modulate protein structure . In this interaction, the lone pair on the oxygen of a peptide carbonyl group
*
overlaps with the antibonding orbital (π ) of the carbonyl group in the subsequent peptide bond. The n*
[3-5]
π interaction was suggested to play roles in several fundamental structural elements in proteins . In
*
[6]
particular, n-π interactions were suggested to stabilize PII conformation . We sought to examine the
*
effects of n→π interactions on PⅡconformation in short model peptides: Ac-aa-NMe2, Ac-aa-OMe
(aa=Ala, Val, Phe, Leu, Ile, Pro). We reason that substitution on the donor or acceptor group would shift
*
*
the electronic delocalization, therefore the strength of an n→π interaction. If n→π interactions indeed
stabilize PⅡ conformation, population of PⅡ will shift upon changing the strength of the interaction;
observation of no effect on PⅡ population with changing strength of the interaction would reveal that
*
*
n→π interactions have neglectable effects on PⅡ conformation. Our data show that the n→π interactions
can stabilize PⅡ conformation. Reference: [1] Shi, Z. S.; Chen, K.; Liu, Z. G.; Kallenbach, N. R. Chem.
Rev. 2006, 106, 1877−1897. [2] Jia-Cherng Horng; Raines, R. T. Protein Sci. 2006, 15, 74-83. [3] Hinderaker,
M. P.; Raines, R. T. Protein science. 2003, 12, 1188-1194. [4] Choudhary, A.; Gandla, D.; Krow, G. R.; Raines,
R. T. J. Am. Chem. Soc. 2009, 131, 7244–7246. [5] Bartlett, G. J.; Choudhary, A.; Raines, R. T.; Woolfson, D.
N. Nature chemical biology, 2010, 6, 615-620. [6] Hodges, J. A.; Raines, R. T. Org. Lett. 2006, 8, 4695–4697
POST 03-303
The Secretion And Expression Of A Series Of B Domain Truncated Recombinant Coagulation Factor
VIII In Hepatocyte
Zhang Jun, Zhu Chong-yang, Zhang Qian-ying, Lin Ming-ming, Wen Quan, Li Xin
Department of cell biology & genetics, Chongqing Medical University, Chongqing, China
The B domain of the coagulation factor (FVIII) is believed to be important for processing, intracellular
transport and secretion of FVIII protein, but the specific regions for these functions still remains
mysterious so far. The investigation aims to preliminarily determine these regions by comparing the
secretion and expression of five B domain truncated variants with full-length wild type FVIII (WT FVIII).
Firstly, six recombinant vectors were contrusted and transiently expressed in hepatocyte (HL-7702 cell
line) that don’t express endogenous FVIII. Media and cell lysates were collected after 48 hours following
tranfection. The secretion and expression of the five B domain truncated variants were analyzed in
comparison to wild type FVIII construct. All six recombinant FVIII constructs were highly expressed in
hepatocyte without significant differences at mRNA level. But the levels of FVIII antigens (FVIII: Ag) in
media exhibited markedly differences for the four recombinant constructs with truncated regions of 840950aa, 950-1200aa, 1200-1400aa and 1400-1642aa respectively. Only one variant 751-840aa truncated
recombinant FVIII demonstrate equivalent relative antigen level, which suggested that the region of 740840aa in B domain seems not to be involved with the FVIII protein transport and extracellular secretion
process. All the remaining regions of B domain could, more or less, be associated with the
posttranslational transport or secretion and the inferred number of glycosylation sites in each truncated
202
POSTER ABSTRACTS
region show no obvious correlation with the abilities of FVIII protein transport and secretion. Key
words: Coagulation factor FVIII; expression; secretion; B domain truncated constructs
POST 03-304
Crystal Structure Studies Of Dipeptidyl Aminopeptidase BII From Pseudoxanthomonas Mexicana
WO24.
Yasumitsu Sakamoto1, Yoshiyuki Suzuki2, Ippei Iizuka1, Chika Tateoka1, Saori Roppongi1, Mayu Fujimoto1,
Koji Inaka3, Hiroaki Tanaka4, Mika Masaki5, Kazunori Ohta5, HIrofumi Okada2, Takamasa Nonaka1, Yasushi
Morikawa2, Kazuo T. Nakamura6, Wataru Ogasawara2, Nobutada Tanaka6
1
Department of Structural Biology, Iwate Medical University, Yahaba, Iwate, Japan, 2Department of
BioEngineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan, 3Maruwa Foods Inc.,
Yamatokoriyama, Nara, Japan, 4Confocal Science Inc., Chiyoda-ku, Tokyo, Japan, 5Japan Aerospace
Exploration Agency, Tsukuba, Ibaraki, Japan, 6School of Pharmacy, Showa University, Shinagawa-ku,
Tokyo, Japan
Dipeptidyl aminopeptidase cleaves a penultimate positioned peptide bond from the NH2 terminus of
peptides. Some asaccharolytic pathogens are known to utilize peptides or protein as a carbon/energy
source. Therefore, these microorganisms secrete a variety of peptidases that involved in peptide
metabolism. Especially, Porphyromonas gingivalis is known to utilize dipeptides, instead of free amino
acids. Thus, dipeptidyl aminopeptidases are very important from the point of view of dipeptides
production. The peptidase family S46 that contains dipeptidyl aminopeptidase BII (DAP BII)
from Pseudoxanthomonas mexicana WO24 is the only exopeptidase family in clan PA peptidases. DAP BII
exhibits a broad specificity for hydrophobic and basic amino acids on P1 position of substrate
peptide. Other enzymes that belong to this family are DPP7 and DPP11 from Porphyromonas gingivalis,
DPP11 from Porphyromonas endodontalis (periodontal pathogen) and DPP11 from Shewanella
putrefaciens (multidrug resistance associated opportunistic pathogen). Moreover, as family S46
peptidases are not found in mammals, we expect our study will be useful for the discovery of specific
inhibitors to family S46 peptidases from these pathogens. Our present phylogenetic and experimental
studies indicated that the catalytic triad of DAP BII is composed of His 86, Asp 224 and Ser 657 and
implied that unknown large helical domains involved in exopeptidase activity[1]. However, threedimensional structure of family S46 peptidases has not yet been reported. Thus, the crystal structure of
DAP BII is essential not only to understand the catalytic mechanism of family S46 peptidases but also to
clarify the structural origin of the exo-type peptidase activities of these enzymes. Recently, we successfully
crystallized DAP BII and collected X-ray diffraction data to 2.3Å resolution from the crystal. This crystal
belongs to space group P212121, with unit-cell parameters a = 76.55, b = 130.86, c = 170.87Å . Crystal
structure analysis by the multi-wavelength anomalous dispersion method is in progress [2]. Here, we
report the first crystallization and structural analysis of DAP BII from P. mexicana WO24 as family S46
peptidases. [1] Suzuki et al., Scientific Reports, 4 4292 (2014) [2] Sakamoto et al., Scientific
Reports, 4 4977 (2014)
POST 03-305
Protein Recognition by Multivalent Fluorescent Molecular Sensors
David Margulies
Organic Chemistry, Weizmann Institute, Rehovot, Israel
Fluorescent molecular sensors have become valuable tools in the analytical biosciences owing to their
high sensitivity and their ability to track proteins in their native environment. A major limitation in using
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POSTER ABSTRACTS
these probes, however, is the lack of a general and easily applicable method for providing them with high
selectivity and high signal-to-noise ratios. In addition, many of these sensors are designed according to
the "lock and key" paradigm; therefore, they cannot be used to analyze biomolecule combinations. In our
1group, we aim to address these problems by developing novel classes of fluorescent molecular sensors.
5
I will present our recent development of fluorescent switches that light up in the presence of specific
enzyme biomarkers, as well as probes that can shed light on analyte combinations in biofluids. In addition,
the design of a sensory system that utilizes both specific and non-specific interactions for distinguishing
between protein isoforms will be discussed.References (1 Rout, B.; Unger, L.; Armony, G.; Iron, M. A.;
Margulies, D. Angew. Chem. Int. Ed. 2012, 21, 12477. (2) Rout, B.; Milko, P.; Iron, M. A.; Motiei, L.;
Margulies, D. J. Am. Chem. Soc 2013, 135, 15330. (3) Rout, B.; Motiei, L.; Margulies, D. Synlett, 2014, In
press, DOI 10.1055/s-0033-1340639. (4) Motiei, L.; Pode, Z.; Koganitsky, A.; Margulies, D. Angew. Chem.
Int. Ed. 2014, In press, DOI: 10.1002/anie.201402501R201402501 (5) Unger-Angel, L.; Rout, B.; Motiei, L.;
Margulies, D. 2014, Submitted.
POST 03-306
Assessing Student Understanding of Foundational Concepts of Protein Structure and Function
Ellis Bell
In recent years there have been a number of reports and publications emphasizing the importance of
student understanding of foundational concepts and core competencies in the context of both
disciplinary and interdisciplinary content and skills. For biochemistry and molecular biology the content
knowledge, skills and allied fields necessary for the discipline have been discussed in a series of recent
papers. Protein Structure and Function was identified as one of the five essential content areas that
students must understand. In the current work, principles of scientific teaching and backward design have
been used to align six critical components of understanding protein structure function relationships with
focused assessment of student understanding and potential teaching strategies. Of the six, two align with
key features of understanding structure (Bonding & Dynamics), two with understanding biological
function (Catalysis & Regulation) while two (Interactions & Evolution) clearly bridge the other concepts.
Integrated with these core concepts of protein structure and function are the essential interdisciplinary
concepts of Modularity, Energy, Change over time, Stochasticity and the Use of Mathematical models.
Using a modified Bloom’s taxonomy Potential assessments aligned to each of the six foundational
concepts of protein structure and function are discussed. In addition to assessing student understanding
of the foundational concepts of protein structure and function the goal of such assessments is to
incorporate key aspects of both the allied fields and the skills necessary for student success in
biochemistry and molecular biology. Finally. potential student centered teaching strategies
for introductory, gateway or capstone courses are suggested. This work was funded by NSF RCN-UBE
Grant 0957205 - Ellis Bell, Principal Investigator
POST 03-307
Dietary Condensed Tannins And Their Effect On Microbial Protein And Theoretical Methane Yield
In Ruminal Fermentation On Cattle In The Northern Mexico
Ericka Fabiola Luisillo-Quiñones2, Gerardo A. Pámanes-Carrasco1, 2, Christian A. Hernández-Vázquez2,
Zaira J. Romo-Astorga2, Esperanza Herrera3
1
UPIDET, Technological Institute of Durango, Durango, Mexico, 2Ingeniería en Tecnologías Ambientales,
Universidad Politécnica de Durango, Durango, Durango, Mexico, 3FMVZ - UJED, Durango, Durango,
Mexico
204
POSTER ABSTRACTS
The purpose of this research was to evaluate the effect of dietary condensed tannins (CT) on microbial
protein, volatile fatty acids (VFA) and theoretical methane yield in rumen. Four rumen fistulated steers
were used in a duplicated 4x4 Latin square design in 21-d adaptation periods. Measured dietary CT were
-1
62.93, 77.11 and 114.92 µg g of DM intake for T1, T2 and T3, respectively. Steers were fed twice daily and
ruminal samples were obtained before feeding (0h) and every 4h until 12h. Methane proportion
(mmol/mol VFA) was calculated from acetate, propionate and butyrate concentrations. Ruminal pH had
no significant differences (p<0.05) between treatments. Significant differences were found in microbial
protein between treatments, where T3 presented the lowest value (952.32 µg CE/mL Ruminal Content)
and T1 the highest value (1471.51 µg CE/mL Ruminal Content). Lower concentrations of acetate,
propionate and butyrate (441.06, 275.67 and 31.96 mmol/mol VFA, respectively) were found in T3 at
8h. No significant differences (p<0.05) were found in acetate/propionate ratio between treatments. The
lowest theoretical CH4 concentration was found in T3 (135.45 mmol/mol VFA) while the highest was found
in T2 (188.57 mmol/mol VFA). Experimental results showed that when low dietary CT is fed, VFA
production decreased, affecting CH4 production and methanogenic bacteria activity without affecting
acetate/propionate ratio and ruminal pH.
POST 03-308
Regulation and Inter-Domain Communications in C-terminal Src Kinase (CSK)
Sulyman Barkho, Levi C. Pierce, Joseph A. Adams, Patricia A. Jennings
University of California, San Diego, La Jolla, California, US
The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation
and are under the principal control of the C-terminal Src Kinase (Csk). Csk and SFKs share a common
modular design with the kinase domain downstream of the N-terminal SH2 and SH3 domains which
regulate catalytic function and membrane localization. While the functions of interfacial segments in
these multidomain kinases are well-investigated, little is known about how surface sites and long-range,
allosteric coupling control protein dynamics and catalytic function. The SH2 domain of Csk is an essential
component for the down-regulation of all SFKs. A unique feature of the SH2 domain of Csk is the tight
turn in place of the canonical CD loop in a surface site far removed from kinase domain interactions. We
used experimental and computational methods to probe the importance of this difference by
constructing a Csk variant with a longer SH2 CD loop that mimics the flexibility found in homologous SH2
domains. Our data indicate that while the fold and function of the isolated domain and the full-length
kinase are not affected by loop elongation, native protein dynamics that are essential for efficient
catalysis are perturbed. These results underscore the sensitivity of intramolecular signaling and catalysis
to native protein dynamics that arise from modest changes in allosteric regions while providing a
potential strategy to alter intrinsic activity and signaling modulation. Furthermore, advanced
computational studies revealed concerted global motions of the two regulatory domains in Csk. We
observed a significant translocation of the SH3 domain about the small kinase lobe which is not
apparent in the crystal structure. This potentially activating transition is in agreement with our
functional and dynamic experimental studies and may provide a potential mechanism for Csk’s kinase
activation via its SH3 domain.
POST 03-309
Site-Specific Molecular Recognition of Proteins by Synthetic Receptors
Adam Urbach
Trinity University, San Antonio, Texas, US
Cucurbit[n]urils are water-soluble, synthetic macrocycles that bind selectively to aromatic amino acid
residues at the N-terminus of peptides and proteins. Molecular recognition is mediated by the
205
POSTER ABSTRACTS
simultaneous inclusion of the aromatic sidechain within the hydrophobic cavity of the cucurbit[n]uril, and
electrostatic attraction of the N-terminal ammonium and peptide NH groups with the carbonyl oxygens
on the rims of the cucurbit[n]uril. These interactions enable the selective binding of an N-terminal
aromatic residue, such as phenylalanine, versus non-terminal aromatic residues, non-aromatic residues,
and amino acids by factors ranging from 10-10,000-fold in binding affinity. Affinities in aqueous solution
are excellent compared to other synthetic receptors, but the limit of dissociation constants is on the order
of 0.1-1 micromolar. This presentation will describe our recent work that addresses challenges in the
areas of recognizing natural proteins, measuring and controlling catalytic proteolysis, achieving
nanomolar binding affinity, separating native proteins, and processing and protecting peptides through
selective interactions with proteases.
POST 03-310
Structure of Dihydromethanopterin Reductase: Redox Transfer in a Cubic Protein Cage
Dan E. McNamara1, Duilio Cascio2, 3, Julien Jorda3, Cheene Bustos4, Tzu-Chi Wang4, Madeline E. Rasche4,
Thomas A. Bobik5, Todd O. Yeates1, 2, 3
1
Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California,
US; 2Molecular Biology Institute, University of California Los Angeles, Los Angeles, California;
3
Department of Energy Institute of Genomics and Proteomics, University of California Los Angeles, Los
Angeles, California, US; 4Department of Chemistry and Biochemistry, California State University
Fullerton, Fullerton, California, US, 5Roy J. Carver Department of Biochemistry, Biophysics, and
Molecular Biology, Iowa State University, Ames, Iowa, US
Dihydromethanopterin reductase (Dmr) is a redox enzyme that plays a key role in
tetrahydromethanopterin (H4MPT) biosynthesis for use in methylotrophy by archaea and some bacteria.
DmrB is a bacterial enzyme known to catalyze the flavin-dependent reduction of dihydromethanopterin
(H2MPT) to H4MPT, but the mechanistic details have not been elucidated previously. We used X-ray
crystallography to determine the structure of DmrB from Burkholderia xenovorans at a resolution of 1.9 Å.
Unexpectedly, the biological unit is a 24-mer composed of eight trimers forming the corners of a cubic
cage-like structure. Within each trimer, monomer-monomer interfaces contain an active site with two
adjacently bound flavin mononucleotide (FMN) ligands, one deeply buried and tightly bound and one
more exposed, for a total of 48 ligands in the biological unit. Isothermal titration calorimetry indicated a
single high-affinity site for FMN and computational docking suggested that the peripheral site could bind
either the observed FMN (the electron donor for the overall reaction) or the pterin, H2MPT (the electron
acceptor for the overall reaction), in configurations ideal for electron transfer to and from the tightlybound FMN. On this basis, we propose a ping-pong mechanism for DmrB-mediated transfer of reducing
equivalents from FMN to the pterin substrate. Sequence analysis suggested that the catalytic mechanism
is conserved among DmrB homologs in bacteria and partially conserved in archaea, where an alternate
electron donor is likely used. In addition to the mechanistic revelations, the structure of DmrB may assist
in the development of anti-obesity drugs based on targeting human gut microbiota.
POST 03-311
The Linkage Between Folding Cooperativity and Allostery: Glutamate Dehydrogenase
Angela Tata, Grace Kingdom, Ellis Bell
Glutamate Dehydrogenase is a homohexameric enzyme showing complex kinetic and regulatory
properties that involve subunit interactions. Our previous work (ref etc) has demonstrated a role for ligand
induced changes in conformational flexibility in modulating regulatory effects of the enzyme. We have
conducted extensive studies on the effects of ligands on the unfolding of the protein by either chemical
206
POSTER ABSTRACTS
denaturants (Guanidine Hydrochloride or Urea- followed by fluorescence) or heat (thermal denaturation
followed by ellipticity at 222nm). We have developed a quantitative analysis of the cooperativity of the
unfolding process using a 4 parameter sigmoidal equation and use it to demonstrate that ligand
interactions lead to changes in unfolding cooperativity. Furthermore correlation of the effects of various
substrates, cofactors and regulatory ligands on the unfolding process suggest that in general increased
flexibility of the protein is related to the ligand producing enhanced activity while decreased flexibility
results in lower activity. Furthermore we have correlated these effects with the temperature dependence
of not only the parameters of folding but also effects on both cofactor cooperativity and ADP
inhibition/activation and GTP inhibition, followed by initial rate kinetics. This work was supported by NSF
Grant MCB 0448905 to EB
POST 03-312
Hydrogen Exchange Of Disordered Proteins In Living Cells
Austin E. Smith1, Zijian Zhou1, Gary J. Pielak1, 2, 3
1
Chemistry, UNC Chapel Hill, Chapel Hill, North Carolina, US, 2Biochemistry and Biophysics, UNC Chapel
Hill, Chapel Hill, North Carolina, US, 3Lineberger Comprehensive Cancer Center, UNC Chapel Hill, Chapel
Hill, North Carolina, US
A truly disordered protein lacks a stable fold and its backbone amide protons exchange with H2O at rates
predicted from studies of unstructured peptides. Accurate estimations of these exchange rates are
important for determining whether disordered proteins possess small amounts of structure, for
determining if disordered proteins gain structure inside cells and for assessing globular protein stability in
cells. Using two model disordered proteins and the NMR experiment, SOLEXSY, we have shown that
exchange rates in Escherichia coli can match those obtained in buffer and those calculated based on
exchange rates from small, unstructured peptides. The data indicate that predicted rates from model
peptides can be applied to hydrogen exchange studies of globular proteins inside E. coli. Most
importantly, we have shown that protein disorder can persist under physiological conditions and that the
crowded cellular interior need not affect the structure of disordered proteins.
POST 03-313
Assessment of UCH-L3 Substrate Selectivity using Engineered Ubiquitin Fusions with Variable
Linker Lengths
Peter Suon, John J. Love
The Ubiquitin Proteasome System (UPS) is a complex system composed of multiple structural and
functional elements that play key roles in cellular processes such as signal transduction, cell cycle
regulation, apoptosis, and protein degradation. Proteins destined for degradation via the proteasome are
first ‘tagged’ with the protein ubiquitin, which is covalently attached to internal lysine residues in the
targeted protein. The enzyme Ubiquitin Carboxy Hydrolase L3 (UCH-L3) is believed to prepare ubiquitin
for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the
ubiquitin C-terminus. Previously in our laboratory, protein substrates of UCH-L3 were engineered and
used to characterize UCHL-3 substrate selectivity. The engineered substrates consisted of N-terminal
monoubiquitinated test variants derived from Streptococcal protein G (Gβ1) and Staphylococcal protein A
(SpAB). The thermal denaturation temperatures (Tm) of the fusion proteins were measured using circular
o
dichroism and span a range of over 60 C. More importantly the rate of hydrolysis for the fusions was
demonstrated to be directly correlated to the Tm of the test variant fused to the C-terminus of ubiquitin.
The engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain
any ‘linker’ residues between the C-terminus of ubiquitin and the N-terminus of the test protein. To
207
POSTER ABSTRACTS
explore the effects of linker length on UCH-L3 hydrolysis we are engineering new UCH-L3 substrates that
contain an unstructured 13 amino acid linker between ubiquitin and the test protein. The thermal stability
of these new fusion protein substrates will be measured using circular dichroism spectroscopy (CD) and
UCH-L3 hydrolysis rates characterized. Our goal is to continue to use these engineered substrates to
explore the mechanics of UCH-L3 and its potential role in protein trafficking and degradation within living
cells.
POST 03-314
Identification, Characterization, and Modification of Fatty Acid AlkylEsterases Found in
Staphylococcus aureus
Benjamin D. Saylor, John J. Love
Alternative energy is a major field of current research. Biodiesel, a mixture of fatty acid alkyl esters, is the
most versatile biofuel in current use. This is due to the fact that it is similar enough to gasoline that it is
compatible with the diesel engines found throughout the existing global infrastructure. Biodiesel
precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. However, the
standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water,
greatly reducing the net energy gained in its manufacture. Our work constitutes an attempt to explore
enzymatic synthesis of biodiesel with lipids like those derived from emerging fuel crops.
Previous
literature describes fatty acid alkyl ester formation in Staphylococcal lesions, formed by partially
characterized esterase activity from an unidentified source. We have identified the enzymes responsible
for this activity by using a combination of size exclusion chromatography, gas chromatography-mass
spectrometry, and mass spectrometric protein sequencing. These two highly similar enzymes in the
glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous
conditions at or near room temperature. We have demonstrated that other, similar lipases do not exhibit
this behavior. We have expressed these Staphylococcal esterases in E. coli, and shown via gas
chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. Based on
sequence similarity to homologous proteins that have already been crystallized, we have predicted a
structure for these enzymes and have engineered mutants with higher rates of catalysis.
Poster Session: Protein Degradation
POST 04-315
Apoptotic Protein Bax is Regulated By The Ubiquitin-Proteasome Pathway
Kwang-Hyun Baek, So-Ra Kim, Jin-Ok Kim
Department of Biomedical Science, CHA University, Seongnam, Korea, Republic of
Bax, one of B-cell lymphoma 2 (Bcl-2) family members, is an important protein in apoptosis signaling
pathway and exists as a cytosolic monomer in unstressed cells. But, when the stress induces cell death
signal, Bax is dimerized and translocated to the outer membrane of the mitochondria, where it induces
the apoptosis by secreting cytochrome c. Cytochrome c is a main protein that initiates apoptosis signaling
pathway. Recently, Bax was identified to be ubiquitinated. Ubiquitination is a death signal of protein, and
ubiquitinated proteins are degraded by the ubiquitin-proteasomal pathway (UPP). Other previous reports
showed that ubiquitin-mediated proteasomal degradation system is present in the outer membrane of
mitochondria. In order to demonstrate the relationship between Bax and deubiquitinating enzymes
208
POSTER ABSTRACTS
(DUBs), we performed the yeast two hybrid screening and immunoprecipitation (IP) analyses. As a result,
we revealed that USP-Bax interacts with Bax, and regulates the stability of Bax through its
deubiquitinating activity. Taken together, we suggest that USP-Bax is a specific DUB for Bax, which may
efficiently induce apoptosis and provide a new therapeutic target for various cancers.
POST 04-316
Discovery and Characterization Of Small Molecule Fragments That Bind And Inhibit the Ubiquitin
Specific Protease 7 (USP7)
Paola Di Lello5, Terry Crawford4, Kurt Deshayes3, Joy Drobnick4, Jake Drummond1, James Ernst2, Lorna
Kategaya3, Michael Kwok2, Cuong Ly4, Till Maurer5, Jeremy Murray5, Chudi Ndubaku4, Rich Pastor4, Lionel
Rouge5, Structural Biology Expression group5, Vickie Tsui4, Ray Zhao4, Kerry Zobel3, Ingrid Wertz3
1
Genentech, South San Francisco, California, US, 2Protein chemistry, Genentech, South San Francisco,
California, US, 3Early Discovery Biochemistry, Genentech, South san francisco, California, US, 4Discovery
Chemistry, Genentech, South san francisco, California, US, 5Structural Biology, Genentech, South San
Francisco, California, US
The tumor suppressor p53 is a transcription factor that induces cell cycle arrest or apoptosis in response
to a variety of cellular stresses, including DNA damage, hyper-proliferation, and oncogenic activation. In
the majority of human cancers the p53 pathway is inactivated by either direct mutations in the p53 gene
or alterations in the pathways that regulate p53 levels. In cancer types that retain wild type p53,
reactivating the p53 pathway is considered a promising strategy for cancer therapy. The Ubiquitin Specific
Protease 7 (USP7) is part of the complex network of proteins that regulates the activity of p53. Recently
USP7 has emerged as an attractive oncology target because its inhibition stabilizes p53, thereby
promoting p53-dependent apoptosis in cancer cells. Our main goal was to identify small molecule
inhibitors of USP7 that could be used as tool compounds in studies aimed at demonstrating the antitumor effects of USP7 inhibition. We screened the Genentech fragment library by Nuclear Magnetic
Resonance (NMR) and discovered a variety of small molecule fragments that bind the catalytic domain of
USP7 in distinct sites. Among these, we found one fragment series that specifically binds USP7 in the
catalytic site and two additional scaffolds that, although binding USP7 in a region adjacent to the catalytic
site (the palm region), inhibit USP7 enzymatic activity. The fragments binding in the palm region appear
to inhibit USP7 by interfering with Ubiquitin binding.
POST 04-317
Polyglycine Hydrolases Secreted by Pathogenic Fungi
Todd A. Naumann2, Donald T. Wicklow2, Todd J. Ward2, Michael J. Naldrett1, Neil P. Price3
1
Donald Danforth Plant Science Center, St. Louis, Missouri, US, 2Bacterial Foodborne Pathogens and
Mycology Research Unit, USDA-ARS-NCAUR, Peoria, Illinois, US, 3Renewable Products Technology
Research Unit, USDA-ARS-NCAUR, Peoria, Illinois, US
Pathogens are known to produce proteases that target host defense proteins. Here we describe
polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the
polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were purified
from two fungal pathogens, Bipolaris zeicola (Bz-cmp) and Epicoccum sorghi (Es-cmp). Both proteases
were shown to cleave three different corn (Zea mays) chitinase substrates. These substrates have
interdomain polyglycine linkers with as many as 14 consecutive glycines. MALDI-TOF MS analysis of
peptide products indicated that polyglycine hydrolases cleave multiple peptide bonds within the
polyglycine linker regions. The peptides produced and their abundance varied with both protease and
substrate. Removal of the amino-terminal 29 amino acids from substrate chitinases resulted in loss of
209
POSTER ABSTRACTS
activity. This suggests that polyglycine hydrolases recognize the short, amino-terminal domain of targeted
chitinases through exosite interactions. To identify these novel proteases, a draft genome sequence was
generated from E. sorghi genomic DNA and purified Es-cmp was subjected to LC-MS/MS peptide
sequencing. Two candidate proteases were identified. We are cloning the cDNAs and creating
heterologous expression strains to produce recombinant proteins. Recombinant proteins will be tested for
the ability to cleave chitinase polyglycine linkers. Our description of polyglycine hydrolase activity
improves understanding of how proteases can evolve to target specific proteins.
POST 04-318
Higher-order Assembly Architecture of the AAA+ Protease Lon Reveals a New Regulatory
Mechanism For Substrate Specificity
Tejas Kalastavadi1, 2, Ellen Vieux1, Breann Brown1, Tania Baker1, 2
1
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts,
US, 2Howard Hughes Medical Institute, Cambridge, Massachusetts, US
Various environmental stresses can result in the accumulation of unfolded, misfolded or otherwise
damaged proteins in cells. Organisms across all kingdoms of life, including bacteria have developed an
extensive protein quality control network that includes holdases, disaggregases, refolding chaperones,
and proteases to combat these insults. The AAA+ Lon protease has been shown to degrade proteins to
regulate cell growth and clear misfolded and damaged proteins during stressful growth conditions.
Historically, Lon has been characterized as a ring-shaped homohexamer. However, we recently reported
biophysical and structural evidence that Lon also assembles into a dodecamer and that this architecture
represents is second active conformation of the protease. Importantly, he activities of the Lon hexamer
and dodecamer vary in an interesting manner; the two enzyme forms degrade some classes of substrates
with equal efficiencies whereas with other classes of substrates the activities differ by more the ten-fold.
We are dissecting the molecular bias of this change in substrate performance by enzyme assembly and
determining the impact of the two assembly states of degradation pathways in vivo. To investigate how
assembly of the Lon protease into the dodecamer modulates substrate choice and flux through the
V217A/Q220A
VQ
enzyme, we used a mutant, lon
(encoding Lon ) that predominantly assembles into a
dodecamer and profiled its substrate specificity in vivo and in vitro. We report that degradation of large
VQ
oligomeric substrates, such as the small heat shock proteins IbpA/IbpB, is impaired with Lon , compared
to the wild type enzyme. In contrast, degradation of small monomeric proteins was essentially unaffected
VQ
by the VQ mutation, with Lon and wild-type Lon having similar degradation profiles for this substrate
class. Thus, we conclude that assembly of the Lon dodecamer from Lon hexamers alters the substrate
selection properties of Lon. This shift in substrate specificity/degradation by Lon oligomerization, which in
turn depends on the intracellular enzyme concentration and perhaps allosteric activators of Lon may
explain the physiological importance of higher-order assembly of Lon protease and its specific roles in
responding to cellular stress conditions.
POST 04-319
A Robust Assay for Protein Unfolding By AAA+ Molecular Machines
Vladimir Baytshtok2, Tania A. Baker2, 1, Robert T. Sauer2
1
HHMI, Massachusetts Institute of Technology, Cambridge, Massachusetts, US, 2Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts, US
AAA+ family molecular machines play critical roles in ATP-dependent protein degradation, which is an
essential process in all living organisms. A typical AAA+ protease consists of a hexameric AAA+ unfoldase
that utilizes the energy of ATP binding and hydrolysis to unfold and translocate protein substrates into a
partner peptidase, which cleaves the polypeptide into small fragments. In addition, some AAA+
210
POSTER ABSTRACTS
unfoldases have chaperone and unfolding activities independent of their partner peptidases in vivo and in
vitro. These peptidase-independent activities have not been characterized extensively, however, due to a
lack of robust unfolding assays with good dynamic range. We have developed a simple and reliable FRETbased assay, in which unfolding/dissociation of donor-labeled and acceptor-labeled homodimers is the
rate-limiting step in formation of heterodimers, and used it to determine the steady-state unfolding
kinetics of the ClpX and ClpA AAA+ enzymes, both of which partner with the ClpP peptidase. We find that
the unfolding activity of ClpX is greater without than with ClpP present. In contrast, by itself, ClpA is a
relatively slow unfoldase, but ClpP stimulates this activity substantially. We also find that hydrolysis in the
D1 ring of ClpA is much more important for unfolding in the absence of ClpP than in its presence.
Although interactions of ClpX and ClpA with their partner peptidase alters their mechanical activities in
unpredictable ways, our new unfolding assay should allow straightforward determination of the effects of
peptidase binding on a broad range of AAA+ unfolding machines.
POST 04-320
Mitochondrial Lace1 ATPase
Lukas Stiburek, Jana Cesnekova, Josef Houstek, Jiri Zeman
1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
Human Lace1 (Lactation elevated 1) is a homolog of yeast Afg1 (ATPase family gene 1) ATPase with
sequence identity ranging from 41,5 % H. sapiens vs. S. cerevisiae to 88,9 % H. sapiens vs. M. musculus.
The protein consists of ATP/GTP binding P-loop motif and a common five domain structure. The aim of
our work was to perform detailed cell biological characterization of human Lace1 utilizing stable shRNAbased RNA interference approach, proteomics and expression analyses using human embryonic kidney
cell line (HEK293). We found that human Lace1 is a mitochondrially localized integral membrane protein
with an apparent molecular weight of 50 kDa. The prepared stable shRNA LACE1 knockdown HEK293 cell
line showed markedly reduced Lace1 protein levels (<10% of controls). SDS-PAGE western blotting
demonstrated markedly elevated levels of subunit SDHA of respiratory complex II, Cox2, Cox3, Cox4,
Cox5a and Cox6aL subunits of respiratory complex IV, and ATP d subunit of complex V. On the other
hand, the amount of the antiapoptotic factor Bcl-2 was found to be decreased in these cells. Furthermore,
slightly elevated levels of the tumor suppressor p53 were found in LACE1 knockdown cells. Quantitative
2D-PAGE analysis coupled to mass spectrometric identification (MS) showed elevated accumulation of
chaperonine 10 and ATPase F1-alpha. Our work have thus far identified Lace1 as a mitochondrial factor
playing a role in protein turnover of subunits of the oxidative phosphorylation system, possibly confirming
its predicted function as an adaptor protein of mitochondrial proteases. Future experiments will involve
short term downregulation of Lace1 using Stealth siRNA approach as well as co-immunoprecipitation
analyses coupled to MS identification with P-loop mutant Lace1 protein. The work was supported by
Grant Agency of the Czech Republic Project GACR 13-072235 and institutional projects UNCE 204011 and
RVOVFN6465.
POST 04-321
The Structure Of The Human Hybrid Proteasome
Edward Morris1, Fabienne Beuron1, Paula da Fonseca2
1
Division of Structural Biology, Institute of Cancer Research, London, United Kingdom, 2Division of
Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, Cambridge, United Kingdom
The proteasome plays a fundamental role in eukaryotic homeostasis by undertaking the highly controlled
degradation of a wide range of proteins, including key cellular regulators such as those controlling cell
cycle progression and apoptosis. In eukaryotes, proteasomes typically consist of the 20S proteolytic core
211
POSTER ABSTRACTS
associated with capping complexes. The well known 26S proteasome, for example, is composed of the 20S
core with 19S regulatory particles (19S RPs) at each end which serve to recognise and unfold ubiquinated
proteins. However, alternative assemblies also exist such as the hybrid proteasome complexes which have
different classes of cap at each end of the 20S core. The hybrid 19S-20S-PA28 proteasome with a 19S-RP
complex at one end of the 20S core and a PA28 11S complex at the other is thought to be involved in
presenting antigen peptides to the immune system. Here we have analysed the three-dimensional
structure of the 19S-20S-PA28 hybrid proteasome derived from HeLa cells by electron microscopy and
single particle analysis. The resulting structure with a resolution of ~25 Å has sufficient detail to allow the
recognition and docking of the component subunits. Comparison with the structure of the human 26S
proteasome reveals conformational rearrangements of the subunits which may be related to the distinct
function of the hybrid proteasome.
POST 04-322
Are the Precursor Sequences of Thiol Proteases Related to Thiol Protease Inhibitors
Meron Tarekegn, David Harry, Kelsey Kines, Ellis Bell
Thiol proteases play significant roles in organisms from all kingdoms and are often synthesized as
precursor forms (zymogens) that undergo activation by proteolysis. Thiol proteases play significant roles
in biological phenomena as diverse as tooth decay, plant root invasion and immune responses and
differentiation. While a number of protein inhibitors of thiol proteases are found in biology the most
ubiquitous are the cystatin/Stefin family of inhibitors. Using a combination of bioinformatics, homology
modelling and molecular dynamics approaches we have investigated the possibility that the precursor
region of thiol protease precursors are structurally related to the thiol protease inhibitors and form an
auto-inhibitory loop that penetrates the active site of the protease. We have complemented these
computational approaches with direct experimentation using chicken cystatin derivatives (phosphorylated
or truncated) as inhibitors of the model thiol protease, papain. Studies of the conformation of the betaturn-beta region of cystatin that correlates to the auto-inhibitory loop of the precursors suggest the need
for additional components to give a folded structure.
POST 04-323
Thiol Proteases & Thiol Protease Inhibitors in C elegans
Kelsey Kines, David Harry, Meron Tarekegn, Ellis Bell
Thiol proteases and their naturally occurring protein inhibitors play critical roles in a large number of
biological processes including the innate immune system. To initiate studies of thiol proteases, their
substrates and naturally occurring inhibitors in the model organism C elegans we have computationally
Screened C elegans sequence databases for cystatin and stefin like molecules and thiol protease like
molecules. A number of sequences have been identified and appropriate homology models created.
Using these models we have looked at potential interactions using various docking routines. To
experimentally explore expressed proteases and their inhibitors in C elegans we have fractionated using
gel filtration and ion exchange chromatography C elegans extracts assaying for general protease activity
and thiol protease activity specifically using assays in the presence and absence of small molecule thiol
protease inhibitors. To investigate potential targets for cystatin-like or stefin-like thiol protease inhibitors
we have use a histidine tagged cystatin as “bait” for thiol proteases. These approaches are being used
212
POSTER ABSTRACTS
with C elegans under a variety of “stress” conditions to explore potential involvement of thiol proteases
and their inhibitors in stress response.
POST 04-324
Structural Analysis of Poly-SUMO Chain Recognition by RNF4-SIMs Domain
Chia-Hsiuan C. Kung, Mandar T. Naik, Hsiu-Ming Shih, Tai-huang Huang
Academia Sinica, Taipei, Taiwan
The E3 ubiquitin-protein ligase RNF4 contains four tandem SUMO interacting motif (SIM) repeats for
selective interaction with poly-SUMO modified proteins, which it targets for degradation. We employed a
multifaceted approach to characterise structures of the RNF4-SIMs domain and tetra-SUMO2 chain to
elucidate the interaction between them. In solution, the SIMs domain was intrinsically disordered and the
linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of RNF4-SIMs domains bind to SUMO2 in
the groove between the β2 strand and the α1-helix parallel to the β2 strand. SIM2 and SIM3 bound to
SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding.
SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is
comparable to that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2/RNF4SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that
the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped
around the SUMO protamers in a superhelical turn without imposing steric hindrance on either
molecule.
POST 04-325
Misfolded Proinsulin Retrotranslocation for Proteasome-dependent Degradation in the Cytosol Can
Be Modulated by Altering the Endoplasmic Reticulum Lumenal Composition
Pen-Jen Lin
Western University of Health Sciences, Pomona, California, US
Accumulation of misfolded proinsulin results in stress in the endoplasmic reticulum (ER) of pancreatic ßcells and is considered the leading cause of certain types of diabetes mellitus (Scheuner D 2008; Schnell
2009). Recent biochemical and genetic evidence has suggested that misfolded proinsulin
retrotranslocates from the ER lumen to the cytosol and is degraded by the proteasome. Such evidence
suggests at the therapeutical potential of increasing the ER-associated protein degradation (ERAD) of
misfolded proinsulin to prevent ß-cell failure (Kammoun 2009; Goeckeler 2010). As a first step to evaluate
the therapeutical potential, we have developed de novo approaches to directly monitor ERAD of
proinsulin in a real time manner. Our results demonstrate that the rate constant of wild type proinsulin
-6
-1
-4
-1
retrotranslocation increased from 6 x 10 · s to 6 x 10 · s after a misfolding-prone mutation occurred,
indicating that misfolded proinsulin is a preferred substrate for ERAD. Moreover, we found that both the
rates of misfolded proinsulin retrotranslocation and degradation increased by 2 fold after the
concentration of the ER lumenal proteins was elevated. These results suggest that ERAD of proinsulin can
be modulated and that the soluble proteins within the ER are responsible for targeting misfolded
proinsulin for degradation.
213
POSTER ABSTRACTS
Poster Session: Frontier High-Throughput Techniques
POST 05-326
Virotrap: Abducting Protein Complexes from Mammalian Cells
Sven Eyckerman, Kevin Titeca, Emmy Vanquickelberghe, Annick Verhee, Noortje Samyn, Delphine De
Sutter, Evy Timmerman, Kris Gevaert, Jan Tavernier
Medical Protein research, VIB -UGent, Gent, Belgium
Affinity purification of protein complexes followed by mass spectrometry is a well-established strategy for
the characterization of these protein assemblies. A critical part in this workflow is the homogenization of
the cells followed by purification, typically through extensive washing of the protein complex, often
resulting in an interaction network biased towards strong(er) interactions. In Virotrap, we exploit the
spontaneous particle formation initiated by expression and multimerization of the HIV-1 GAG protein.
Specific protein complexes are sorted and trapped inside these particles by fusion of a bait protein to
GAG. Using a novel purification protocol we demonstrate that Virotrap allows the detection of known
binary interactions, as well as the identification of new interactions by mass spectrometry. Virotrap thus
provides a unique addition to the arsenal of protein interaction technologies by omitting the need for cell
homogenization.
POST 05-327
Global Measurement Of Protein Localization In C. elegans With Tissue And Subcellular Specificity
Aaron Reinke, Eric Bennett, Emily Troemel
UCSD, La Jolla, California, US
Organisms must adapt dynamically throughout development, and respond to external perturbations such
as infection with pathogens. These responses ultimately manifest themselves as differences in both
protein levels and localization, but these changes have been difficult to measure in multicellular
organisms in a global and specific manner. To comprehensively measure changes in protein levels and
localization, we have adapted a technology for use in the model multicellular organism C. elegans that
allows for proteins in both specific tissues and cellular compartments to be tagged with a chemical
handle in vivo. This approach relies on the localized expression of ascorbate peroxidase (APX) from
soybean that can activate a biotin-phenol substrate in the presence of H2O2, which results in proteins in
the close proximity of the enzyme being covalently modified with biotin (Rhee et al. Science 2013). These
proteins can then be purified and measured using mass spectrometry. We have shown that this approach
specifically tags proteins in multiple subcellular locations and tissues and the identified proteins are highly
enriched for proteins known to be expressed in those compartments and tissues. Differences between
nuclear and cytoplasmic localized proteins in multiple tissues at larval and adult stages are currently being
investigated, which will provide a view of protein dynamics throughout development. Additionally, we
have investigated the response to infection with microsporidia, which is a naturally occurring intracellular
intestinal pathogen of C. elegans. Worms expressing cytoplasmic or nuclear localized APX in the intestine
were infected with microsporidia and proteins were tagged with biotin and purified. The identities of
these proteins were determined using mass spectrometry. 89 microsporidia proteins were identified,
including three large families of novel proteins (one predicted to be secreted and the other two predicted
to be localized to the pathogen membrane). In our mass spectrometry studies, the large secreted family
was identified as being in both the cytoplasm and nucleus, whereas the large membrane-bound families
were restricted to the cytoplasm. We are currently extending this approach to other intestinal pathogens
to localize other pathogen proteins that are used to manipulate the host, as well as to measure how the
host proteome is remodeled during infection.
214
POSTER ABSTRACTS
POST 05-328
Broad Scope and Coverage of Functionally Relevant Groups for Sequences in the Enolase
Superfamily
Brian Westwood1, Stacy Knutson3, Janelle Leuthaeuser1, Patricia Babbitt2, Jacquelyn Fetrow3
1
Molecular Genetics and Genomics, Wake Forest University School of Medicine, Winston-Salem, North
Carolina, US, 2Department of Bioengineering and Therapeutic Sciences and California Institute for
Quantitative Biosciences, University of California at San Francisco, San Francisco, California, US, 3Physics
and Computer Science, Wake Forest University , Winston-Salem, North Carolina, US
Automated methods of comparing full sequential or global structural similarities are often used to transfer
functional annotation to uncharacterized sequences. Manually curated databases overcome many pitfalls
of generalized similarity transfer by focusing on the fine molecular detail of enzymes at their active site,
assembled from experimental characterization of mechanistically determinant residues found in the
literature. A reliable high-throughput method of grouping proteins with discrete molecular function
based on sequence motifs in the active site is needed to keep pace with the rate of protein sequence
information accumulation. We have previously developed a two level iterative protocol for clustering
structurally characterized proteins based on their functional microenvironments, which utilizes the Deacon
Active Site Profiler (DASP) to define enzyme clusters based on active site profile
characteristics. Subsequent searches of GenBank NR using functionally relevant groups from the enolase
superfamily result in discrete groups of significantly scoring sequences (core hits). We find good
correlation between these search results and classifications in the gold standard Structure-Function
Linkage Database (SFLD). While there was appreciable cross hitting at less significant scores (i.e.
sequences identified were not all unique to their functionally relevant group profile searches), these noncore hits identify sequences from groups of enolases which had no structural representatives. This
discretization of scores and nested iterative searching of core and non-core hits allows us to increase the
scope and coverage of sequences in the enolase superfamily, over the naïve structurally derived
functionally relevant group profiles alone.
POST 05-329
A Molecular Toolkit For Single Molecule Peptide Sequencing
Jim Havranek, Ben Borgo
Genetics, Washington University of St. Louis, St. Louis, Missouri, US
The advent of next-generation DNA sequencing has revolutionized molecular biology, ushering in a ‘postgenomic’ era. Inspired by the relentless miniaturization and parallelization of sequencing technologies
that have enabled this revolution, we have begun development of a toolkit of molecules that we hope will
enable a massively multiplexed, single molecule peptide sequencing assay inspired by Edman
degradation. We envision an iterative procedure in which the N-terminal amino acids of immobilized,
spatially segregated individual peptides are identified by a series of binding probes. Subsequently, each
N-terminal amino acid is removed, regenerating a new terminus for another round of identification. While
nature has provided the protein tools that are needed for sequencing DNA, to realize our goal of
next-generation peptide sequencing we must engineer our own. For our strategy to succeed, we require
a set of recognition domains capable of distinguishing the naturally occurring amino acids, an enzymatic
activity for cleaving N-terminal residues, and a single-molecule detection platform. We have assembled a
set of amino acid recognition domains and characterized their binding affinities for peptides harboring
each of the natural amino acids (except cysteine) at their N-termini. We developed a probabilistic
framework for converting from binding events to an amino acid ‘read’. We used computer-aided protein
design to construct a novel enzyme that accomplishes the cleavage step of Edman degradation in
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POSTER ABSTRACTS
aqueous conditions and neutral pH. Finally, we discuss the prospects for transitioning our molecular
toolkit to a single molecule protein detection platform.
POST 05-330
Novel Solubility Fusion Partners High Throughput System to Produce Soluble Proteins
Saurabh Sen, Eric Steinmetz, Sally Floyd, David Mead, Mark Maffitt
Lucigen Corporation, Middleton, Wisconsin, US
A large fraction of heterologous proteins are insoluble or poorly expressed in Escherichia coli. One
solution to this problem is to fuse a “solubility tag” to the target protein. Selection of the best tag is a
time consuming trial-and-error process that requires testing multiple different promoters, strains, and
cloning technologies. Lucigen has developed a simple solution to simultaneously test multiple tags within
the context of a single promoter, vector and host system. Lucigen’s Solubility Panel consists of 24
cleavable fusion partners within a robust enzyme-free cloning platform. In addition, a novel yellow
fluorescent protein, LucY, significantly enhances solubility and expression while providing an instant visual
report of the amount of soluble, active protein. This system permits rapid, simultaneous screening of
multiple factors demonstrated to improve solubility and/or expression in a high throughput format. Using
difficult targets, e.g. LRRK2 (the kinase for Parkinson's Disease) and others have proven successful with
this new solubility suite.
POST 05-331
Elucidating The Global Metabolic Regulatory Role Of Prokaryotic Enzyme Post-Translational
Modifications Through Systems Biology Analysis
Nathan E. Lewis1, Roger Chang2, Chen Yang4, Hooman Hefzi3, Bernhard Palsson3, George Church2
1
Pediatrics, University of California, San Diego, La Jolla, California, US, 2Harvard Medical School, Boston,
Massachusetts, US, 3Bioengineering, University of California, San Diego, La Jolla, California,
US, 4Shanghai Institutes for Biological Sciences, Shanghai, China
For decades it has been asserted that relatively few prokaryotic enzymes are regulated by posttranslational modifications (PTMs). However, recent proteomic studies have discovered a wealth of PTMs
in prokaryotes, especially with respect to metabolic enzymes. It is not known what these PTMs may do in
prokaryotes. To elucidate the detailed biochemical functions of these PTMs and how they influence E.
coli physiology, we developed a high-throughput approach that integrates proteomic data, metabolic
network analysis, protein structure analysis, and targeted genome engineering. Through this approach we
demonstrate that these PTMs play an important role in rapidly regulating metabolism to cope with
fluctuations in the nutritional microenvironment of the cell. Specifically, we use a novel metabolic pathway
modeling method, called Regulated Metabolic Branch Analysis (RuMBA), to identify enzymes that should
require metabolic regulation in response to noise from fluctuating metabolite concentrations. We show
that PTMs are particularly enriched among these enzymes and complement known allosteric regulatory
mechanisms. Furthermore, regulated PTM sites are highly conserved and located near enzyme active sites.
We further elucidate detailed mechanisms by which these PTMs regulate flux by integrating RuMBA with
enzyme assays, protein structure analyses, and screens of PTM mutants generated by multiplexed
automated genome editing technologies. Through this we show that PTMs are employed far more than
previously anticipated to regulate prokaryotic metabolism in response to intrinsic and extrinsic metabolic
noise. Furthermore, we show that by integrating disparate data types with systems biology analyses,
detailed biochemical biochemistry can be predicted and one can prioritize confirmatory biochemical
assays.
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POSTER ABSTRACTS
In this study disparate data types were integrated using systems biology analyses in order to predict the
biochemical mechanisms through which specific phosphorylation and acetylation events regulate
enzymes in E. coli.
Poster Session: Membrane Proteins & Receptors
POST 06-332
What’s on the Menu: Identification of the Hydrocarbon Transport Systems as a first step in Marine
Oil-Degradation by Alcanivorax borkumensis
Swapnav Deka1, Chad Brautigam2
1
Plano East Senior High School, Plano, Texas, US, 2Biophysics, UT Southwestern Medical Center, Dallas,
Texas, US
Alcanivorax borkumensis is a marine hydrocarbon-degrading bacterium that has shown to play an
important role in the biological removal of petroleum hydrocarbons from oil-spills and polluted marine
waters. Bacterial biodegradation of hydrocarbon takes place in the interior of the cell (cytoplasm) and
requires the passage of water insoluble (hydrophobic) oil molecules across the cell membranes. However,
no hydrocarbon transporter in A. borkumensis has been revealed from its genome sequencing. Therefore,
the purpose of the current study was to employ both computational biology (bioinformatics) and
microbiological tools to search for such transporter genes. In the bioinformatics, various means were used
to identify the presence of genes encoding the channel proteins in A. borkumensis. These analyses led to
a significant discovery of both outer- (Abo_0193) and inner- (Abo_0687) membrane putative hydrophobic
transporters in A. borkumensis. The three-dimensional models of the transporter proteins (Abo_0193 and
Abo_0687) further supported their hypothetical roles as potential hydrocarbon transporters. The ability to
transport chain-like oil-field hydrocarbons was demonstrated by the growth ofA. borkumensis under
various hydrocarbons. Based on these findings, a “transport model” for oil-like hydrocarbon from the
extracellular environment to the cytoplasm has been developed as a first step in biodegradation. To verify
the proposed model along with the chemical nature of hydrocarbon binding to the proposed
hydrophobic cavity, we already have crystallized the recombinant Abo_0687. The crystal structure will
enable the better understanding of hydrocarbon transport process and to engineer more
adaptive/efficient oil-eating bacteria for bioremediation.
POST 06-333
Keeping in Touch: T-cadherin Impedes Dissociation of adiponectin Receptor 1 Dimers
Tobias Leimer2, David Kosel2, Karin Mörl2, Barbara Ranscht1, Annette G. Beck-Sickinger2
1
Medical Research Institute, Sanford Burnham, La Jolla, California, US, 2Institute of Biochemistry, Leipzig
University, Leipzig, Germany
Adiponectin and its receptors possess a powerful potential for the treatment of the metabolic syndrome
and other diseases (1). However, the complexity of the receptor-ligand-system limits our understanding
of the molecular mechanism, which is important for pharmaceutical therapies. It is known that AdipoR1
and AdipoR2 are able to form homo- and heteromers which dissociate upon adiponectin stimulation and
seem to have an impact on the signal transduction (1, 2). In addition T-cadherin emerges as a third
adiponectin receptor, which is not only able to bind adiponectin but also able to alter cellular response
of this adipokine (3). Bimolecular fluorescence complementation and flow cytometry analysis were
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POSTER ABSTRACTS
applied to investigate the effect of T-cadherin on AdipoR1 dimerization. AdipoR1 and T-cadherin were
co-transfected in different ratios in HEK293 cells and incubated with adiponectin. The quantification of
the mean fluorescence demonstrates that T-cadherin impedes the adiponectin-induced dissociation of
the AdipoR1 dimers. Consequently, the interaction of the three adiponectin receptors has to be
considered in terms of cellular response and regulation of the adiponectin signaling.
(1) Kosel D,
Heiker JT, Juhl C, Wottawah CM, Blüher M, Mörl K, Beck-Sickinger AG, (2010). Dimerization of
adiponectin receptor 1 is inhibited by adiponectin. J Cell Sci.;123(Pt 8):1320-8. (2) Almabouada F, DiazRuiz A, Rabanal-Ruiz Y, Peinado JR, Vazquez-Martinez R, Malagon MM, (2013). Adiponectin receptors
form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.
J Biol Chem.;288(5):3112-25. (3) Denzel MS, Scimia MC, Zumstein PM, Walsh K, Ruiz-Lozano P, Ranscht
B, (2010). T-cadherin is critical for adiponectin-mediated cardioprotection in mice. J Clin
Invest.;120(12):4342-52.
POST 06-334
A New Rigidity-Based Model For Allosteric Communication In G-Protein Coupled Receptors
Adnan Sljoka1, 2
1
University of Colorado, Boulder, Boulder, Colorado, US, 2Ryerson University, Toronto, Ontario, Canada
Given the 3-D structure of a protein, understanding how it functions depends in critical ways on
predicting which parts are rigid and which are flexible. The rigidity and flexibility analysis of the molecular
graph, using a fast combinatorial rigidity algorithm - the pebble game (implemented in programs FIRST at
flexweb.asu.edu, ProFlex and Kinari), can rapidly decompose a protein into flexible and rigid regions. In
this study we extend this technique and develop a novel computational approach for detecting protein
allosteric interactions. It is widely believed that the binding of a ligand at the allosteric site triggers a
conformational change that is transmitted through the protein to cause a rearrangement and alteration of
the shape of the active site. However, the underlying allosteric mechanism is still not well understood. In
this work we introduce a rigidity-based allosteric mode of communication together with an algorithm
which can detect transmission of rigidity and shape changes between two (or more) distant binding sites
in allosteric proteins. Our algorithm is also used to predict and identify regions in the protein that are
critical for the coupled communication between distant sites (i.e. allosteric pathways). Starting with a set
of known GPCR structures, we apply these methods and show how binding of an activating ligand (i.e.
agonist) triggers small rigidity changes which propagate to the critical G-protein binding regions. In
contrast, in the inactive GPCR structures no such rigidity allosteric communication is observed. Detailed
predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and
results are compared with experimental evidence. These results show that rigidity-based allosteric model
and algorithm is a powerful new tool for detecting allostery in GPCRs. This research was partially funded
by NSERC (Canada).
POST 06-335
Keeping it Simple: The construction of Biologically Active Proteins With Minimal Chemical Diversity
Erin N. Heim1, Jez L. Marston2, Kelly M. Chacon1, Lisa M. Petti1, Daniel DiMaio1
1
Yale School of Medicine, New Haven, Connecticut, US, 2Yale College, New Haven, Connecticut, US
The bovine papillomavirus E5 protein is a 44-amino acid transmembrane protein that transforms
fibroblasts to tumorigenicity by activating the platelet-derived growth factor β receptor (PDGFβr). Unlike
the natural ligand, PDGF, which binds to the extracellular domain of the PDGFβr, the E5 protein activates
the PDGFβr in a ligand-independent manner by binding directly and specifically to the transmembrane
domain of the receptor. Based on this understanding of the E5 protein, we explored the minimal chemical
diversity required to generate an active transmembrane protein. We constructed retroviral libraries
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POSTER ABSTRACTS
expressing short artificial proteins consisting of randomized hydrophobic sequences and used genetic
selection to isolate rare proteins that transformed cells. The simplest proteins we have isolated thus far
are 26-amino acids long and consist of only three different hydrophobic amino acids. Biochemical and
genetic analysis indicates that these proteins activate the PDGFβr by interacting functionally with its
transmembrane domain. The unprecedented chemical simplicity of these sequences challenges our
understanding of the chemical diversity required to generate a biologically active protein. These findings
provide insight into protein-protein interactions, oncogenesis, and protein evolution, and they have
important implications for artificial protein engineering.
POST 06-336
Characterization Of The Second Immunoglobulin Domain (Ig2) Of The Human Receptor
CRTAM/CD355 And Its Role In The Formation Of Dimer
Juan C. Barragan-Galvez1, Luis Brieba-Castro1, Vianney Ortiz-Navarrete2
1
National Laboratory of Genomics for Biodiversity, The Center for Research and Advanced Studies
(CINVESTAV), Irapuato, Guanajuato, Mexico, 2Molecular Biomedicine, The Center for Research and
Advanced Studies of the National, Gustavo Madero, Distrito Federal, Mexico
The recognition of non-self and abnormal self-antigens and the activation of NK cells and CTLs are
mediated by Antigen Receptor Responses (AgRs), a number of costimulator and adhesion molecules
involved in this mechanism. Among the immune costimulatory adhesion proteins, attention has been
focused on the Class I-restricted T cell-associated molecule (CRTAM) or CD355, which is a class I
transmembrane protein and belongs the immunoglobulin superfamily (IgSF), is a marker expressed in
immature thymocytes, and thymus heterophilically interacts with Nectin-like 2 (CADM1) during the early
+
phases of development of CD8 and play role in the formation of cell polarity. CRTAM as an
immunoglobulin molecule having a conserved disulfide bridge in the first variable-like domain (IgV)
between cysteines 38-98 and the other in the second constant-like domain 2 (C2), between cysteines 141196. Zhang and Rubinstein (2013) determined the crystal structure of the first domain IgV and reveal an
interface forming the cis-dimer and interaction with the ligand, however there is no evidence of the role
played by the second domain IgG. Cloning of the extracellular region of CRTAM in the system GST-fusion
expressed in Rosetta-gamihas been successfully purified from the soluble fraction by GST-affinity
chromatography. Our evidence shows that the second IgC domain is responsible for dimer formation in
solution in the absence of the first domain IgV, this according to the chromatographic profiles and
immunoblot assays.
POST 06-337
Expression, Purification And Functional Refolding Of Human Olfactory Receptor Expressed In
Escherichia coli
Heehong Yang1, Sae Ryun Ahn1, Tai Hyun Park1, 2
1
Seoul National University, Seoul, Korea, Republic of, 2Advanced Institutes of Convergence Technology,
Suwon, Republic of Korea
Olfactory receptors (ORs) known as the G-protein coupled receptor (GPCR) family are integral membrane
proteins that have seven transmembrane helices structure. Because of their hydrophobicity and
complicated structure, ORs are difficult to be overexpressed, solubilized and purified. These receptors
bind to their specific odorant molecules, thus its specificity is useful for the application of bioelectronic
nose. In addition, highly purified and well-refolded human olfactory receptor (hOR) has a powerful
advantage and is used to various fields, such as drug screening, protein/ligand interaction and analyzing
the hOR structure. Herein, hOR2AG1 was overexpressed with glutathione S-transferase (GST) at the N-
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POSTER ABSTRACTS
terminus, and 6xHis-tag at the C-terminus as an inclusion body. The fusion protein of hOR2AG1 was
solubilized in buffer containing ionic detergent and applied to metal affinity chromatography for the
purification. GST domain was removed using proteolytic cleavage before the elution from the column.
After the purification, hOR2AG1 was successfully refolded using nonionic detergents and methyl-ßcyclodextrin as protein folding assistants. Finally we obtained the highly purified and well-refolded
hOR2AG1. The functional activity was confirmed by using circular dichroism (CD) spectrum and recording
the quenching of the intrinsic receptor fluorescence on the addition of odorant molecules. This study can
be applied to develop protein-based sensing system including bioelectronic nose and to analyze the
native GPCR structure using solid-state NMR, X-ray crystallography, or neutron scattering.
POST 06-338
The Importance of CD4 Allostericity for Interaction with HIV glycoprotein 120
Nichole M. Cerutti, Vinesh Jugnarain, Alexio Capovilla
Molecular Medicine and Haematology, University of the Witwatersrand, Parktown, Gauteng, South
Africa
Human receptor CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes
where it plays a key role in the activation of immunostimulatory T cells. This function is diverted by the
Human Immunodeficiency Virus (HIV) envelope glycoprotein (gp120), which uses CD4 as its primary
receptor for cell entry. While growing evidence suggests that redox exchange reactions involving CD4
disulphides (potentially catalysed by cell surface-secreted oxidoreductases) play an essential role in
regulating the activity of CD4, their mechanism(s), biological utility and structural consequences that may
be applicable to the designs of novel antiviral therapies and vaccines remain incompletely understood. To
gain more insights into the importance of redox activity in the mechanism of HIV entry, a panel of
recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, were used to
show that Thioredoxin (Trx), an oxidoreductase found on the cell surface, reduces 2dCD4 highly efficiently,
catalysing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulphidelinked 2dCD4 dimer. HIV-1 gp120 was shown to be incapable of binding a fully oxidised, monomeric
2dCD4 in which both domain 1 and 2 disulphides are intact, but binds robustly to reduced equivalents
that are the products of Trx-mediated isomerisation. This Trx-driven dimerisation of CD4, a process
believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation
complexes, is shown to be impaired when CD4 is bound to gp120. Finally, preliminary, low-resolution
structural analysis of individual CD4 domains 1 and 2 are suggestive of intrinsic metastability in domain 2,
and reduction of its resident allosteric disulphide bond likely underpins the structural rearrangements in
CD4 that are required for efficient interaction with gp120. Overall, these findings emphasise the
fundamental importance of redox pathways in the biochemical mechanism of HIV entry, and illustrate the
potential feasibility of exploiting these for the development of novel antiviral ligands.
POST 06-339
Structural Characterization Of The Major Pilin Subunit From The Bacterial Nanowires Of Geobacter
Sulfurreducens
Patrick N. Reardon2, Karl T. Mueller2, 1
1
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania,
US, 2Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland,
Washington, US
Geobacter sulfurreducens and related species of proteobacteria can utilize a wide range of insoluble
extracellular electron acceptors, such as metal oxides, during respiration. These bacteria are widespread
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POSTER ABSTRACTS
in the environment and are important to the cycling of minerals and nutrients. The transport of electrons
by G. sulfurreducens is thought to be facilitated by filamentous type IV pili, often referred to as bacterial
nanowires. Type IVa pili are protein polymers that are comprised of primarily one major pilin protein. We
have recently determined the atomic resolution structure in detergent micelles of the major pilin protein
from G. sulfurreducens, PilA, using high resolution NMR spectroscopy. The protein is >85% α-helical and
is structurally similar to the N-terminal region of other non-conductive type IVa pilins. Modeling of the
PilA NMR structure onto the type IVa pilus fiber model from Neisseria gonorrhoeae provides insight into
the organization of aromatic amino acids that have been proposed to play a role in electrical conduction.
POST 06-340
X-ray Structures Reveal Bent Conformation For All CNTN Family Members
Roman M. Nikolaienko, Samuel E. Bouyain
School of biological sciences, University of Missouri Kansas City, Kansas City, Missouri, US
The family of contactins (CNTNs) includes six highly homologous proteins: CNTN1-6. They are expressed
primarily in the developing and adult neural system. The biological role of CNTNs comprises cell adhesion,
axon guidance, neurite outgrowth and oligodendrocyte maturation. CNTN1, 2, 5 and 6 are expressed at
synapses and available data suggest that they participate in synapse formation and plasticity. All
members of CNTN family are organized into six N-terminal immunoglobulin like (Ig) domains, four
fibronectin type III (FN) domains and a glycophosphatidylinositol anchor. Although structural analyses
demonstrate that the 4 N-terminal Ig domains of CNTNs adopt a horseshoe-like conformation, less is
known about the conformation of the rest of the CNTN ectodomain, and in particular about its FN
region. As a first step to characterize the role of the CNTN FN region, we have determined the crystal
structures of first three FN domains for all six members of CNTN family. Our data show that these protein
fragments adopt an L-shaped conformation with a bend between the second and the third FN domains.
This conformation is consistent with the presence of CNTNs at the synaptic cleft where two apposing
membranes are separated by ~ 20 nm. To further investigate the conformation of the CNTN ectodomain
we have also determined the crystal structure for the Ig5-FN2 region of CNTN3, which adopts an entirely
extended arrangement. Taken together, our structural analyses indicate that CNTNs adopt a conformation
resembling that of synaptic molecules NCAM1 and OCAM, and suggest that such conformations might be
a general attribute of synaptic cell adhesion molecules.
POST 06-341
Visualization of HIV-1 Envelope Glycoprotein In Live Cells By Labeling it with GFP in the gp120
Subunit
Shuhei Nakane2, 1, Aikichi Iwamoto3, Zene Matsuda2, 1
1
China-Japan Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese
Academy of Sciences, Beijing, China, 2Research Center for Asian Infectious Diseases, Institute of Medical
Science, The University of Tokyo, Tokyo, Japan, 3Advanced Clinical Research Center, Division of
Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Human Immunodeficiency Virus type-1 (HIV-1) is a causative agent of AIDS. The envelope glycoprotein
(Env) of HIV-1 consists of non-covalently associated gp120 and gp41, generated from the gp160
precursor. The cytoplasmic tail (CT) of gp41 is about 150 amino acids long and shown to regulate
intracellular trafficking of Env through interactions with cellular proteins. This process has been studied
well; however, most of the observations have been made by immunofluorescence and lack dynamic
information. It has been known that the extension of CT even with a short peptide sequence may affect
Env functions. Indeed, our attempt to attach GFP at the C-terminus of CT decreased Env processing from
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POSTER ABSTRACTS
gp160 into gp120 and gp41. Therefore, we made Envs with GFP-insertion (GFP-Envs) into gp120 variable
loops in codon-optimized HXB2 Env. Among the 5 variable loops (V1-V5), V4 and V5 loops were tolerant
to GFP insertion, and these GFP-Envs showed similar fusion activities to that of the wild type both in cellcell and virus-cell fusion assays. Using these GFP-Envs, we next generated the mutant in which the CT
portion was deleted (ΔCT GFP-Env). Intracellular distribution of ΔCT GFP-Env was compared with GFPEnv in transfected HeLa cells. Both the wild type and ΔCT GFP-Envs showed the similar pattern of
intracellular distributions in ER and Golgi. In addition to this pattern, GFP fluorescence was also
observed as a particle-like appearance. A statistical analysis revealed that ΔCT GFP-Env showed
significantly higher numbers of such particles than the wild-type GFP-Env. These particles were
colocalized with Golgi and endosome markers, but not with ER, lysosome, and peroxisome markers.
Furthermore, these particles of ΔCT GFP-Env were confirmed to move together with vesicles containing
Golgi or endosome markers in live cells, suggesting that these particles were intracellular vesicles
moving between Golgi and plasma membrane. Higher incidence of these vesicles of ΔCT GFP-Env may
support the existence of the previously suggested Golgi retention motif in addition to the internalization
motif in CT that inhibit cell-surface expression of Env. These results show that our GFP-Env is useful for
the dynamic analysis of intracellular trafficking of Env.
POST 06-342
Hemifusion Induced By The HA2 Subunit Of Influenza Virus Hemagglutinin: Respective Major,
Moderate, And Minor Contributions Of The Soluble Ectodomain, Fusion Peptide, And
Transmembrane Regions
Punsisi Upeka Ratnayake
Michigan State University, Lansing, Michigan, US
Influenza virus is enveloped by a membrane and initial infection of a cell includes endocytosis, reduction
of endosomal pH to the 5-6 range, and fusion of viral and endosomal membranes with deposition of the
viral capsid into the cytoplasm. Fusion is catalyzed by the hemagglutinin HA2 subunit protein which is
~210-residue monotopic protein of the viral membrane with ~25-residue N-terminal “fusion peptide” (FP)
that binds the endosomal membrane and ~160-residue “soluble ectodomain” (SHA2) that lie outside the
virus, followed by the transmembrane domain (TM )domain. This project is a comparative study of vesicle
fusion induced by SHA2, FHA2=FP+SHA2,SHA2TM=SHA2+TM and HA2=FP+SHA2+TM+cytoplasmic
domain protein constructs. Rapid vesicle fusion was induced by all constructs with order SHA2,SHA2TM
POST 06-343
Development Of A Quantitative, Real-Time, Label-Free Assay For Ligands Interacting With The HIV
Envelope Glycoprotein In Virus-Like Particles
Jennifer Seedorff, Edward Berger
National Institutes of Health, Bethesda, Maryland, US
Enveloped viruses typically encode membrane-bound proteins to enable entry into target cells. HIV
entry is catalyzed by a trimer of gp120/gp41 heterodimers, referred to collectively as the Envelope
glycoproteins (Env).
Interaction with CD4 and co-receptor (CCR5 or CXCR4) cause allosteric
conformational changes in the trimeric Env, yet most quantitate studies of HIV entry have used purified,
soluble gp120 monomeric constructs. The study of the purified monomeric components involved in HIV
entry has greatly enhanced our understanding of how HIV enters cells and how to neutralize
infection. However, context matters. Results from studies using monomeric, soluble gp120 have not
always translated to the more biologically relevant trimeric Env, as was the case for a failed clinical
candidate, soluble CD4. To improve our understanding of the interactions that occur in the context of
the trimeric Env, we have employed BioLayer Interferometry (BLI) to quantitatively study the
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POSTER ABSTRACTS
interactions between Env and soluble ligands. Virus-like particles (VLPs) were generated using gag/pol
driven budding that incorporate WT Env or control VLPs containing no Env. We found that crude
preparations of VLPs could be captured directly from cell-culture media using biotynlated-Galanthus
Nivalis Lectin that had been coated on streptavidin biosensors. Antibodies of interest could then bind to
the captured HIV Env pseudovirus particles. As expected the Env specific antibodies were unable to bind
empty VLPs that lacked Env. Additionally, an isotype matched control antibody showed no significant
binding to either the empty or Env VLPs. These results demonstrate the specificity of the interaction
between the antibody and Env. We were able to measure both the kinetic and the steady-state
equilibrium of the interaction. The kinetic Kd typically reported a tighter interaction than did the steadystate equilibrium analysis. We believe that this is most likely due to rebinding of the antibody to HIV Env
during the dissociation phase of the experiment. Alternate capture and buffer conditions are being
evaluated for improvements in quantitation, especially of the dissociation phase. The ability to use BLI
to quantitate antibody-membrane protein interactions should be broadly applicable to the study of
other membrane proteins of interest.
POST 06-344
Silicon Transport In Diatoms: The Key To Unlocking Their Full Potential?
Sarah Ratcliffe, Michael Knight, Laura Senior, Paul Curnow
School of Biochemistry, University of Bristol, Bristol, United Kingdom
Diatoms are unicellular algae, found in both marine and freshwater environments, which produce highly
intricate silica shells at the nanoscale. These microscopic organisms have long been of interest to
biologists and biochemists but more recently they are gaining the interest of the much wider scientific
community as their potential in both the global carbon cycle and nanotechnology is becoming
increasingly apparent. Whilst the incredible nanostructured silica cases (frustules) of many species of
diatoms have been repeatedly observed in nature, there is still a lack of understanding as to how they use
silicic acid from their external environment and polymerise it so precisely under such ambient conditions.
Being able to recreate and control this process artificially is of great interest in the fields of engineering,
nanotechnology, pharmacology, and the semiconductor industry, to name but a few. Our research
therefore focuses on the family of silicon transport proteins (SITs) already identified in multiple species of
diatoms with a view to understand their mechanism of action, both individually and collaboratively. In
recent years the group have used yeast as an expression system for SITs from the marine
diatom Thalassiosira pseudonana but this project utilises biolistics to transform diatoms themselves with
one of these SITs (TpSIT1) in order to try and get a better understanding of the biochemistry involved.
Here, I will discuss both the advantages and disadvantages of studying these proteins in the host
organism, and what we hope to achieve by doing so. I will also present the use of Xenopus oocytes in the
study of a wider range of SITs from multiple species of both freshwater and marine diatoms. This could
help provide important information about silicon biomineralisation in diatoms that could be utilised by
many in vitro.
POST 06-345
Mechanistic Studies of the Alternative Complex III from the Photosynthetic bacterium Chloroflexus
aurantiacus
Erica L. Majumder1, Robert E. Blankenship1, 2
1
Chemistry, Washington University in St. Louis, St. Louis, Missouri, US, 2Biology, Washington University
in St. Louis, St. Louis, Missouri, US
The Alternative Complex III (ACIII) is a novel energy-conserving integral membrane protein complex that
functions in the cyclic electron transport chain of Filamentous Anoxygenic Phototrophs and other
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POSTER ABSTRACTS
bacteria. ACIII is predicted to be the functional replacement for mitochondrial complex III,
cytochrome bc1 or b6f complexes, but has no structural homology or evolutionary relationship to these
complexes. The oxidoreductase activity of ACIII has been previously demonstrated, showing the role it
plays in the photosynthetic cyclic electron transport chain. The proposed final step of electron transfer
in the complex, the monoheme ActE subunit, has been studied in vivo and reconstituted with the
protein demonstrating its necessity in oxidoreductase activity. The biggest question regarding ACIII has
been its ability to pump protons across the membrane and contribute to the electrochemical gradient.
Work described here seeks to answer this question by reconstituting ACIII into liposomes and
monitoring for a pH change resulting from proton translocation corresponding to enzymatic activity.
Results will provide evidence if ACIII is a proton pump.
POST 06-346
Structure and Function Of The Diatom Silicon Transporter
Michael Knight, Laura Senior, Sarah Ratcliffe, Paul Curnow
Biochemistry, University of Bristol, Bristol, United Kingdom
Diatoms are eukaryotic algae responsible for the majority of global biosilica formation, fixing > 200TMol
silica annually. However, the mechanism by which they take up silicic acid from the environment is poorly
understood at the molecular level. This project will focus on the recombinant expression, purification and
biochemical characterisation of silicon transporters (SITs) from a number of diatom species. Protocols exist
for the purification of SIT3 from the model diatom T. pseudonana using yeast as an expression system
(Curnow et al, (2012) Biochemistry 51: 3776). We intend to build on this prior work by applying this
method to a number of other silicon transporter homologues in order to identify the best target for
subsequent structural and functional studies. This poster will present preliminary data showing that
promising targets can be identified through small scale expression tests and have been purified under a
range of different conditions. Purified proteins are analysed by biochemical and biophysical techniques
including size exclusion chromatography, circular dichroism, western blotting and fluorescent cysteine
labelling.
POST 06-347
Intermolecular Interactions Between The Intracellular Domains Of Arabidopsis CRINKLY4 (ACR4)
Receptor-Like Kinase And Homologs
Matthew R. Meyer1, Shweta Shah2, Gururaj A. Rao2
1
Dept. of Medicine, Washington University School of Medicine, St. Louis, Missouri,
US, 2Biochem.Biophys. Molec.Biol., Iowa State University, Ames, Iowa, US
Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) involved in the global development of the
plant. ACR4 mutant plants show disorganized epidermal formation in leaves and reproductive tissues
consequentially affecting aerial organ development. Additionally, loss of ACR4 function results in
proliferation of columella stem cells (CSC) in the root tip and increased lateral root initiation in developing
roots. A negative feedback mechanism has been proposed between the CLE40 peptide, ACR4, and the
WOX5 transcription factor to regulate CSC differentiation. The Arabidopsis genome encodes four
homologs to ACR4 that contain sequence similarity and analogous architectural elements to ACR4,
termed Arabidopsis CRINKLY4 Related (AtCRRs) proteins. Genetic and cell biology studies have suggested
potential communication between members of the ACR4 gene family. However, little biochemical
evidence is available to ascertain the molecular aspects of receptor hetero-oligomerization and the role of
phosphorylation in these interactions. Therefore, we have undertaken an investigation of the in vitro
interactions between the intracellular domains (ICD) of ACR4 and the CRRs and an additional RLK involved
in epidermis formation, ALE2. We demonstrate that ACR4 can interact with all four CRRs in a
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POSTER ABSTRACTS
phosphorylation independent manner. Furthermore, sequence analysis of the ACR4 gene family has
revealed a conserved KDSAF motif that may be involved in protein-protein interactions among the
receptor family. We demonstrate that CRR3, CRK1, and ALE2 peptides harboring this conserved motif are
able to bind to the ACR4 kinase domain, presumably through the LLSLL motif in the kinase N-lobe.
POST 06-348
Expression, Purification And Reconstitution Of The Aromatic Acid Transporter PcaK
Christian Pernstich, Laura Senior, Katherine A. MacInnes, Marc Forsaith, Paul Curnow
+
The aromatic acid:H symporter family of integral membrane proteins play an important role in the
microbial metabolism of aromatic compounds. Here, we show that the 4-hydroxybenzoate transporter
from Acinetobacter sp. ADP1, PcaK, can be successfully overexpressed in Escherichia coli and purified by
affinity chromatography. Affinity-purified PcaK is a stable, monodisperse homotrimer in the detergent ndodecyl-β-D-maltopyranoside supplemented with cholesteryl hemisuccinate. The purified protein has αhelical secondary structure and can be reconstituted to a functional state in synthetic proteoliposomes.
Asymmetric substrate transport was observed when proteoliposomes were energized by applying an
electrochemical proton gradient or a membrane potential but not by ΔpH alone. PcaK was selective in
transporting 4-hydroxybenzoate and 3,4-dihydroxybenzoate over closely related compounds, confirming
previous reports on substrate specificity. However, PcaK also showed an unexpected preference for
transporting 2-hydroxybenzoates. These results provide the basis for further detailed studies of the
structure and function of this family of transporters.
POST 06-349
Insight Into Receptor-Active Conformation Of Apolipoprotein E Revealed By XL-MS: New Clues For
A Putative Importance Of Helix 4 Elongation In Receptor Recognition.
Nicolas Henry2, Stéphanie Deroo2, Florian Stengel1, Eva-Maria Krammer2, Rouslan Efremov3, Guy
Vandebussche2, Martine Prevost2, Ruedi Aebersold1, Vincent Raussens2
1
Inst. f. Molekulare Systembiologie, ETH, Zürich, Zürich, Switzerland, 2Laboratory of Structure and
Function of Biological Membranes, ULB, Brussels, Brussels, Belgium, 3Department of Structural Biology
(VIB), VUB, Brussels, Brussels, Belgium
Apolipoprotein E is a 34 kDa protein involved in lipid transport and cholesterol homeostasis within the
plasma and central nervous system. Upon binding to lipoprotein particles, apoE adopts its active
conformation allowing it to bind cell surface receptors such as the low density lipoprotein receptor
(LDLr). This interaction mediates the clearance of apoE containing lipoproteins through an endocytosis
pathway and therefore reduces plasma cholesterol levels, explaining the strong anti-atherogenic effect
of apoE. Despite numerous studies undertaken recently to unravel the structure-function relationship
underlying this activation mechanism, no high resolution model has been provided yet. In addition, the
apoE4 isoform is known to be the major risk factor in the development of late-onset Alzheimer’s
disease. Our work focuses on the structural characterization of apoE at the surface of disc-shaped
lipoproteins (nanodiscs). Using optimized conditions, we were able to separate a single homogeneous
and stable population of apoE:POPC nanodiscs as shown by electron microscopy. ApoE molecules at the
surface of the nanodiscs have been successfully cross-linked. The resulting cross-links have been
identified by mass spectroscopy and further used as distance constraints to build 3D models of the
active conformation of apoE in the presence of lipid (POPC) molecules. These models were submitted to
a 50 ns molecular dynamic. The new presented model(s) provide(s) important structural data at the
amino acid level. We noticed tertiary and secondary structure changes in the presence of lipids that may
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POSTER ABSTRACTS
trigger the receptor-active conformation of apoE. Of the utmost interest, the elongation of helix 4 (the
helix containing amino-acids interacting with the LDLr) redefines key residue positions in close proximity
and in the right orientation with regard to the well-defined receptor binding region (aa 136-150) of
apoE. This approach, based on cross-linking assisted by mass spectrometry (XL-MS) identification and
combined to computational chemistry, is a successful alternative to protein structure determination not
suitable for NMR or x-ray crystallography studies, in particular when no high resolution protein structure
is already available and in the presence of lipids.
POST 06-350
The Little Lipid That Could: Elucidating the Effects of Small Amounts of Phosphatidic Acid on
the α-Synuclein Membrane Interaction
Sara K. Hess, Jennifer C. Lee
National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, US
Understanding how environmental factors affect α-synuclein (α-syn) conformation is critical because its
misfolding has been implicated in Parkinson's disease etiology. The role of membranes in α-syn
conformation is of particular interest because membranes alter the protein-folding landscape and are
ubiquitous in vivo. At physiological pH, α-syn binds tightly to equal molar phosphatidylcholine (PC) and
phosphatidic acid (PA) large unilamellar vesicles (LUVs), but weakly to those made of equal molar PC and
phosphatidylserine (PS); however, in synaptic vesicles, PS is the predominant anionic lipid while PA
comprises < 2% of the lipids in these vesicles. We discovered that physiological amounts of PA “doped”
into LUVs composed of PC, PS, and phosphatidylethanolamine (PE), a prevalent phospholipid in vivo, can
greatly enhance α-syn binding at neutral pH as seen by circular dichroism spectroscopy detecting αhelical structure formation. Current work is focused on investigating this special relationship between αsyn and PA, as well as the role of PE, by time-resolved fluorescence and lipid reorganization experiments.
POST 06-351
Structure-based Analysis of Protein Modifications in G Protein Signaling – A New Approach
to Prioritize PTMs in a Protein Complex
Henry Dewhurst, Shilpa Choudhury, Matthew P. Torres
Biology, Georgia Institute of Technology, Atlanta, Georgia, US
G protein signal transduction is one of nature’s most widely utilized and evolutionarily conserved
molecular mechanisms of converting extracellular stimuli into intracellular responses across the plasma
membrane of cells. G protein signaling (GPS) proteins, including 7-transmembrane receptors (GPCRs),
heterotrimeric G proteins (Gαβγ) and Regulators of G-protein Signaling (RGS) proteins function as
members of dynamic complexes and are subject to regulation by post-translational modifications
(PTMs). The vast majority of existing PTM data is now acquired without protein bias using mass
spectrometry-based approaches, but lacks functional context necessary to understand the biological
implications. An emerging hypothesis is that the observation frequency of any site-specific PTM reflects
a propensity for structural specificity that can reveal undiscovered mechanisms of protein regulation. To
identify the structural topology of PTM “hot spots” in GPS proteins we systematically compiled all
experimentally validated GPS protein PTMs from all available database resources and across all
eukaryotes. A total of 2,266 unique PTMs were coalesced across GPCR, Gα, Gβ, Gγ and RGS proteins.
Using a novel in-house PTM analysis algorithm called Structural Projection of PTMs (SPoP), each PTM
was automatically projected onto crystal structures representing the heterotrimer in the inactive as well
as receptor-activated states. PTM hot spots were visualized by colored frequency projection and the
data were filtered by solvent accessibility and location within protein-protein interfaces to reveal the
subset of PTM hotspots with regulatory potential (Figure). More than 20 PTM hotspots were identified,
226
POSTER ABSTRACTS
many of which are located within catalytic, regulatory or subunit interface structures. Most hot spots
within heterotrimeric G proteins are also previously unreported sites of biological regulation by PTMs.
To determine the degree to which the PTM topology was evolutionarily conserved we used affinity
purification mass spectrometry (AP-MS) to identify PTMs on yeast heterotrimeric G proteins. We
discovered several previously unknown phosphorylation sites under receptor activation or nutrient
stress conditions – revealing overlap with the SPoP PTM topology. We conclude that SPoP enables the
structural analysis of PTM topologies in GPS proteins, revealing new potential sites of G protein signal
regulation.
POST 06-352
Molecular Characterization of the Arabidopsis CRINKLY4 receptor-like kinase (ACR4) Intracellular
Domain Coupled With Transmembrane Domain
Shweta Shah1, Matthew R. Meyer2, Gururaj Rao1
1
Roy J. Carver Dept. of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames,
Iowa, US, 2Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, US
Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase is required for overall growth and development of
the plant. ACR4 architecture possesses an extracellular ligand binding domain, a transmembrane (TM)
helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) a core kinase domain
(KD) and a C-terminal (CTD) domain. Previously, we have characterized the ICD of ACR4 and provided
some mechanistic insights into molecular interaction between JMD and KD (Meyer et al. Biochemistry.
2011, 50(12), 2170; Meyer et al. Arch Biochem Biophys. 2013, 535(2), 101). However, the molecular
association of the TM domain with plasma membrane and its effect on the functioning of ICD domain is
still poorly understood. Some recent studies suggest that the ACR4 kinase TM domain plays a critical role
in forming homo and heteromeric complexes in a membrane-bound environment (Stahl et al. Curr Biol.
2013, 23(5), 362-71). The main objective of this study is to characterize ACR4 ICD while still associated
with the TM domain. Towards this end, we have recombinantly expressed a protein with N-terminal 6HSUMO tag and the TM domain followed by ICD (SUMO-TM-ICD). In spite of the hydrophobic nature of
the TM domain, we were able to obtain soluble and active protein. Size exclusion chromatography
experiments suggest that the protein is a monomer even in the absence of detergent. Although the
protein was soluble in the absence of detergent, it showed enhanced kinase activity in the presence of
Tween 20 and Triton X100. Further, the protein phosphorylation sites have also been mapped. In order to
mimic the natural membrane bound environment, we have prepared SUMO-TM-ICD nanodiscs. The
SUMO-TM-ICD nanodiscs were characterized by transmission electron microscopy and were found to be
around 20 nm in size. SUMO-TM-ICD nanodisc remains functionally active and affords great potential for
further characterization of SUMO-TM-ICD in its near-native environment.
POST 06-353
Improved Fusion Protein Strategies For Crystallization Of G-protein Coupled Receptors
Yi Zheng, Ling Qin, Lauren Holden, Chunxia Zhao, Tracy Handel
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La jolla,
California, US
Protein fusion is instrumental for the recent breakthrough in the crystallization and structure
determination of GPCRs, which are important drug targets. Not only has the fusion partner increased
receptor yield and stability it also provides flexible crystal contacts for crystallization. However, finding the
right fusion aid that leads to the final structure is still a matter of trial and error and very time consuming.
In particular the fusion strategy for the intra cellular loop 3 (ICL3) is to systematically slide the fusion
protein within the ICL3 staring from a middle position. Depending on the length of the ICL3 and the
227
POSTER ABSTRACTS
number of potential fusion candidates it will be a substantial increase of the fusion constructs to be
screened. In order to speed up the effort we compared all the GPCR structures with ICL3 fusion
determined so far and found a correlation between linker lengths and structure resolutions. Therefore we
propose a rational design for the ICL3 fusion, which resulted in successful fusion constructs for three
different chemokine receptors with meaningful improvement of yield and stability.
POST 06-354
Biochemical Characterization Of Interaction Between The Arabidopsis Receptor-like kinase (ACR4)
and phosphatase, PP2A3.
Priyanka Sandal1, Matthew R. Meyer2, Petra E. Gilmore2, Ried Townsend2, Aragula G. Rao1
1
Roy J Carver Department of Biochemistry,Biophysics and Molecular Biology, Iowa State University,
Ames, Iowa, US, 2Medicine, Washington University School of Medicine , St Louis, Missouri, US
Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase that is required for the global development of the
plant and is mechanistically involved in controlling the transition between formative and proliferative cell
divisions in the roots (De Smet et al., 2008). We have previously described some biochemical properties of
the intracellular kinase domain of ACR4 and characterized the autophosphorylation sites (Meyer et al.,
2011; 2013). Genetic studies in Arabidopsis have now identified PP2A3, a member of the serine/threonine
PPP phosphatase family, as a downstream interacting protein of ACR4. To complement the inplanta findings, we have expressed the catalytic subunit of Arabidopsis PP2A3 (PP2A3c) in E.coli and
purified the His-tagged protein under denaturing conditions using 8M urea. Proper folding of the
renatured protein was ascertained by circular dichroism. Phosphatase activity was determined using a
molybdate dye assay with a synthetic phosphopeptide substrate. The interaction of PP2A3c with
the E.coli expressed intracellular domain of ACR4 was demonstrated by pull down assay, gel–filtration
studies and far western blots. In-vitro assays demonstrated that PP2A3c was in fact a substrate for ACR4
kinase and was phosphorylated on at least seven sites as determined by mass spectrometry. However, the
activity of PP2A3c as a protein phosphatase against phosphorylated ACR4 remains ambiguous. It is known
that the catalytic subunit of PPP phosphatases is highly (~90%) conserved throughout eukaryotes with
characteristic sequence motifs, reactive-site residues and a conserved C-terminal leucine residue.
Importantly, a fully active phosphatase is a hetero-trimeric holoenzyme composed of scaffold A,
regulatory B and a C subunit with a carboxy-methylated leucine. We suggest that the absence of A and B
subunits, and the inability of E.coli expression system to post-translationally modify the C subunit, might
explain the absence of phosphatase activity of PP2A3c against ACR4. Our future experiments will
therefore be conducted with PP2A subunit proteins expressed in Baculovirus
POST 06-355
The Proteomic Signature Of The Leukolike Vector Unveils The Presence Of Molecules Able To
Improve Self-Tolerance Of The Drug Delivery Systems
Claudia Corbo2, 1, 3, Alessandro Parodi2, 3, Roberto Molinaro2, Michael Evangelopoulos 2, David A. Engler 4,
Shilpa Scaria 2, Francesco Salvatore1, Anthony C. Engler 4, Ennio Tasciotti2
1
CEINGE, Advanced Biotechnologies, Naples, Italy, 2Department of Nanomedicine, The Houston
Methodist research institute, Houston, Texas, US, 3SDN, Fondazione IRCCS, Naples, Italy, 4Proteomics
Programmatic Core Laboratory, Houston Methodist Research Institute, Houston, Texas, US
Nanomedicine aims to improve the therapeutic effects of conventional drugs by increasing their
biocompatibility and targeting while decreasing the potential adverse side effects. In order to provide
nanocarriers with these features, many surface modification strategies based on antibodies, polymers,
and peptides were developed. However, multiple surface modifications increase the complexity of the
synthesis process and very often result inefficient in simultaneously avoiding immune response
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POSTER ABSTRACTS
activation while targeting sick tissues. Bio-inspired approaches are currently being investigated to
enable synthetic particles with multiple therapeutic capabilities. LeukoLike vectors (LLV) are nanoporous
silicon particles coated with purified leukocyte membranes. LLV were previously shown to inhibit
immune system recognition and efficiently target the inflamed endothelium that often characterizes
the site of the disease [1]. Here we characterized the protein composition of the coating by highthroughput proteomic analysis and the results, validated by Western Blotting assays, revealed the
presence of self-tolerance biomarkers and targeting receptors on the nanoparticle’s surface. Among
them CD45, CD47 and MHC-I were identified as key players in determining LLV biocompatibility, while
Leukocyte Associated Function-1 (LFA-1) and Mac-1 contributed to the LLV targeting ability and
bioactivity towards inflamed endothelium. We analyzed the whole data set of proteins that can be
purified from leukocyte membranes and consequently, transferred onto the silicon surface, thus
demonstrating an enrichment of leukocyte membrane proteins, i.e. more than 70% of identified
proteins were plasma membrane-associated proteins. The use of cell membranes to prevent particles
body clearance and improve targeting abilities represents a new paradigm shift for the development of
safe and efficient drug delivery platforms. References Parodi A. et al. Synthetic nanoparticles
functionalized with biomimetic leukocyte membranes possess cell-like functions. Nature
Nanotechnoly. 2013. 8(1): 61-8..
POST 06-356
Crystal Structure of phosphate-bound V1-ATPase of Enterococcus hirae
Kano Suzuki2, Kenji Mizutani2, Yoshiko Ishizuka-Katsura1, Takaho Terada3, Mikako Shirouzu1, Shigeyuki
Yokoyama3, Ichiro Yamato4, Takeshi Murata2
1
Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama,
Kanagawa, Japan, 2Graduate School of Science, Chiba University, Chiba-shi, Chiba, Japan, 3Structural
Biology Laboratory, RIKEN, Yokohama, Kanagawa, Japan, 4Department of Biological Science and
Technology, Tokyo University of Science, Katsushika-ku, Tokyo, Japan
In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as ATP-dependent proton
pumps. They are composed of a hydrophilic domain (V1) and a membrane-embedded ion-transporting
domain (VO) and peripheral stalks connecting V1 and VO. V1 is a rotary motor, which is composed of a
hexagonally arranged catalytic A3B3 complex and a central axis DF complex that rotates using ATP
hydrolysis energy. Enterococcus hirae V-ATPase acts as a primary ion pump similar to eukaryotic VATPase. We have reported crystal structures of the nucleotide-free and AMP-PNP-bound A3B3 complex,
and those of the nucleotide-free and AMP-PNP-bound V1-ATPase. They are asymmetric structures, and
1
we proposed the rotation mechanism . In order to understand when V1-ATPase releases Pi, which arises
by ATP hydrolysis, we obtained the crystal structures of A3B3 and V1 with Pi. The A3B3 and DF complexes
were expressed using Escherichia coli cell-free protein expression system and purified. V1 (A3B3DF) was
purified by gel filtration after incubation of A3B3 with an express concentration of DF. Crystals of A3B3 and
V1 grew in sitting drops by vapor diffusion. Diffraction data were collected and the structures of A3B3 and
V1 were solved by molecular replacement using the crystal structure of nucleotide-free A3B3 (PDB ID code
3VR2) and AMP-PNP-bound V1 (PDB ID code 3VR6), respectively. The structures of A3B3 in 10 mM Pi and
V1 in 2 mM Pi were determined at 2.60 Å and 2.89 Å, respectively. These overall structures are similar to
those of nucleotide-free structures. There is a strong electron density for Pi:Mg in the binding site of V1,
whereas the Pi is not observed in the nucleotide-binding sites of A3B3. We would like to discuss the
hydrolysis mechanism of V-ATPase by comparing with the solved V1-ATPase structures. 1. Satoshi Arai, et
al. (2013) Nature, 493, 703-7
POST 06-357
229
POSTER ABSTRACTS
Structural Analysis for the Interaction of sialic T antigen glycopeptide of HSV-1 with Entry Receptor
PILRα
Takao Nomura4, Jiro Sakamoto3, 2, Fumina Oosaka4, Kosuke Kakita3, 1, Atsushi Furukawa4, Masahiro
Anada1, Shunichi Hashimoto1, Kimiko Kuroki2, Toyoyuki Ose2, Hisashi Arase5, Takashi Saitoh4, Katsumi
Maenaka4, 2
1
Laboratory of Synthetic and Industrial Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido
University, Sapporo, Hokkaido, Japan, 2Laboratory of Biomolecular Science, Faculty of Pharmaceutical
Sciences, Hokkaido University, Sapporo, Hokkaido, Japan, 3Graduate School of Life Science, Hokkaido
University, Sapporo, Hokkaido, Japan, 4Center for Research and Education on Drug Discovery, Faculty of
Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan, 5Department of Immunochemistry,
Research Institute for Microbial Diseases, Osaka University, Osaka, Osaka, Japan
Herpes simplex Virus-1 (HSV-1) is a pathogenic virus which causes various diseases, such as encephalitis, a
cutaneous disease, and child herpes. Paired immunoglobulin-like type 2 receptors (PILRs) are expressed
broadly on immune-related cells. PILRs are a member of paired receptor family, and have high homology
extracellular region, but can transmit opposite signal depending on inhibitory (PILRα) or activating (PILRβ)
motifs in the intracellular region. Glycoprotein B (gB) of HSV utilizes PILRa as an entry receptor, while, gB
could not bind to PILRβ. However, the molecular mechanism of the interaction between PILRα and gB is
not well understood. Recently, we have revealed that glycosylated peptide in gB is involved in the
interaction with PILRα (Kuroki et al. PNAS (2014) in press). This glycopeptide was composed of sialyl T
antigen (sTn) and proline-containing peptide. We determined the crystal structure of PILRα complexed
with sTn glycopeptide. This complex structure supports functional data that PILRα recognizes both sugar
and peptide. Isothermal titration calorimetry and surface plasmon resonance measurements showed sTn
glycopeptide bind to the PILRα with several μM. In order to analyze the structural change of PILRα
mediated by the interaction with the glycosylated peptide, we carried out the NMR titration experiments
15
onto N labeled PILRα with the glycosylated peptide, non-glycosylated peptide and sialic acid. The large
chemical shift change on some amino acid residues were observed with the glycosylated peptide,
although little chemical shift change was observed in non-glycosylated peptide and sialic acid. These
results indicated that the both sTn antigen and peptide sequence in the glycoprotein could play an
important role in the recognition of PILRα. We will discuss the infection mechanism and the rational drug
design for the therapeutic treatment for HSV-1 infection.
POST 06-358
Structural Snapshots of the α-helical Pore-forming Toxin FraC Reveal The Molecular Basis Of Its
Activation In Membranes.
Koji Tanaka, Jose M. Caaveiro, Kouhei Tsumoto
Dept. of Engineering, The Univ. of Tokyo, Tokyo, Japan
Folding and assembly of α-helical membrane proteins is a fundamental question in biology. In spite of
the enormous effort exerted, knowledge of the structural basis of their folding and assembly is limited
to a handful of model proteins due to technical challenges. The α-pore forming toxins (α-PFTs) are
cytolytic proteins that bind to cellular membranes, where spontaneously transform from a watersoluble form to α-helical transmembrane pores. The massive metamorphosis of α-PFTs thus provides a
framework for studying assembly of α-helical membrane proteins. Fragaceatoxin C (FraC) is a suitable αPFT secreted by the sea anemone Actinia fragacea. In this study we have investigated the structural
transition of FraC by determining its crystal structures in the water-soluble and lipid-bound monomeric
forms, and that of an assembly intermediate. The lipid-bound form of FraC was obtained by crystallizing
the protein with 1,2-dihexanoyl-sn-glycero-3-phosphocholine, a water-soluble phospholipid. A single
protein binds up to four lipids, which is to the best of our knowledge the first crystal structure of a
monotopic protein bound to multiple lipids. Comparison of ten independently lipid-bound protein
230
POSTER ABSTRACTS
chains obtained in different space groups allowed us to propose a mechanism of binding of FraC to
membranes. In addition, comparison of the monomeric form with the assembly intermediate suggested
that unfolding of the N-terminal transmembrane domain starts the metamorphosis from monomer to
transmembrane pore. We are also working on the preparation of crystals and processing x-ray
diffraction data of the transmembrane pore of FraC. In summary, multivalent protein-lipid and proteinprotein interactions in the context of the membrane trigger the spontaneous conformational change
and assembly of a soluble protein into a multitopic membrane protein.
POST 06-359
Membrane Proteins Bind Lipids Selectively To Modulate Their Structure And Function
Eamonn Reading, Art Laganowsky, Timothy M. Allison, Carol V. Robinson
University of Oxford, Oxford, United Kingdom
Previous studies have established that the folding, structure and function of membrane proteins are
influenced by their lipid environments and that lipids can bind to specific sites, for example in potassium
channels. Fundamental questions remain however regarding the extent of membrane protein selectivity
toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of
lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large
conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IMMS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of
membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank
bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein
structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting
comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its
proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids with cardiolipin
imparting the most significant resistance to unfolding. Subsequently, through functional assays, we
discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly
selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like
environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound,
reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer.
Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events
enabling distinction of lipids that merely bind from those that modulate membrane protein structure
and/or function. We anticipate that these findings will be influential not only for defining the selectivity of
membrane proteins toward lipids but also for understanding the role of lipids in modulating function or
drug binding.
POST 06-360
NMR Studies of G-Protein Coupled Receptors
Jasmina Radoicic, Sang Ho Park, Anna De Angelis, Bibhuti Das, Sabrina Berkamp, Stanley J. Opella
UCSD, La Jolla, California, US
G protein coupled receptors (GPCRs) are a class of integral membrane proteins composed of seven
transmembrane (TM) helical domains that are involved in a number of essential biological processes
ranging from signal transduction to cell proliferation and angiogenesis. CXCR1 is the high affinity receptor
of interleukin-8 (IL8), which is a major mediator of inflammatory and immune responses, including cancer.
Many methods of structure determination require significant modifications to be made to the protein of
interest, resulting in an environment that is far from the native phospholipid bilayer. Rather than
modifying the protein or its environment, we tailor our techniques to suit the native properties of
membrane proteins. We have determined the structure of the unmodified, wild type receptor in
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POSTER ABSTRACTS
phospholipid bilayers using rotationally aligned (RA) solid state NMR techniques and are in the process of
further refining the structure, including the sidechains, with the ultimate goal of determining the structure
of the unmodified CXCR1-IL8 complex to better understand the signal transduction process. Studies have
also been done looking at interactions between the receptor and its associated G-protein as well as drug
binding studies with potential therapeutics.
POST 06-361
Structure Determination of Vpu from HIV-1 by NMR
Hua Zhang, Eugene Lin, Stanley Opella
The viroporin protein “u” (Vpu) is an 81-residue membrane protein encoded in HIV-1 genome. It consists
of a transmembrane (TM) domain and a cytoplasmic (Cyto) domain, which are associated with different
biological activities that contribute to the pathogenicity of HIV-1 infections in humans. Vpu TM interacts
with the human immuno restriction factor BST-2 as an antagonist to enhance the release of newly formed
virus particles from the infected cells. Recently it has also been discovered to interact with NK cell receptor
NK, T-cell, B-cell antigen (NTB-A) to induce down-modulation of NTB-A and prevent HIV-Infected cells
from degranulation and lysing by NK cells. The Vpu-Cyto removes CD4 receptor from the ER and causes
its subsequent degradation. It is essential to determine the three-dimensional structure of Vpu in order to
obtain an understanding of its molecular mechanisms. A combination of solution and solid-state NMR
experiments are used to obtain protein structural information from different constructs of Vpu. Vpu-Cyto
is prepared in aqueous media, isotropic DHPC micelles and DMPC nanodisks; and the structures in
different conditions are determined base on distance and angular restraints obtained from paramagnetic
relaxation enhancement (PRE) experiments and residue dipolar coupling (RDC) measurements. Solid-state
NMR techniques are employed to study proteins in lipid bilayer, which is a more biologically relevant
environment than micelles. A novel MAS experiments are being implemented to study Vpu full-length
15
13
1
15
1
13
incorporated into DMPC liposomes. N, C chemical shift as well as H- N, H- C dipolar couplings can
be measured and converted to equivalent structural restraints. Structural features of Vpu obtained from
the combination of various techniques will be presented. In addition, we also use solution NMR HSQC
chemical shift perturbation method to map the specific binding site in both Vpu TM and NTB-A TM, and
reveal the molecular mechanism of the interaction.
POST 06-362
NMR Studies of a GPCR with Ligand Bound: CXCR1 and Interleukin-8
Sabrina Berkamp, Anna De Angelis, Bibhuti Das, Sang Ho Park, Mitchell J. Zhao, Jasmina Radoicic, Stanley
J. Opella
G-protein coupled receptors (GPCRs) are the largest family of transmembrane receptors in eukaryotes.
Although there are an increasing number of structures of these membrane proteins being determined by
X-ray crystallography and NMR spectroscopy, little is known about the structures of the active, ligand
bound states. We are currently studying the chemokine receptor CXCR1. It has only one high-affinity
ligand: the chemokine interleukin 8 (IL-8). Although a complete structure of a chemokine receptor with
ligand bound is currently lacking, it is known that the N-terminal part and a loop of the chemokine bind
the N-terminus of the receptor. Mutational and other biochemical studies have shown that the
extracellular loops of the GPCR form a secondary binding site of lower affinity. IL-8 can dimerize, but the
monomeric form is the high-affinity ligand. We have studied the interaction of IL-8 with the N-terminal
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POSTER ABSTRACTS
domain of CXCR1 (ND), and have mapped the binding site of ND to a monomeric form of IL-8 and
compared this to the binding site of wildtype, dimeric IL-8. Using a construct consisting of only the Nterminal domain and the first transmembrane helix of CXCR1 (1TM) we have shown that IL-8 binds to the
receptor in a 1:1 molar ratio and that binding is pH dependent. We are currently working on solving the
structure of IL-8 bound to 1TM as well as full-length CXCR1 using rotationally aligned solid state NMR
spectroscopy and oriented sample NMR spectroscopy, and progress will be presented.
POST 06-363
Membrane Protein Folding Stability and Kinetics in Bilayers
Yu-Chu Chang1, 2, James U. Bowie1, 2
1
Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, US, 2UCLA-DOE Institute for
Genomics and Proteomics, Los Angeles, California, US
The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then
extrapolated back to zero denaturant conditions to obtain unfolding free energies under native
conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the
denaturants may have complex effects on the membrane or micellar structure. We therefore sought to
measure stability under native conditions using a method that does not perturb the properties of the
membrane or membrane mimetics. We employ a technique called steric trapping to measure the
thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin
has a high thermodynamic stability, with an unfolding free energy of ~11 kcal/mol in di-myristoylphosphotidylcholine (DMPC) bicelles. Nevertheless, the stability is much lower than predicted by
extrapolation of measurements made at high denaturant concentrations. We investigated the
discrepancy and found that unfolding free energy is not linear with denaturant concentrations, so long
extrapolations of helical membrane protein unfolding free energies must be treated with caution. We
therefore believe these are the first measurements of thermodynamic stability of a large helical membrane
protein under native conditions. We also extend steric trapping system to membrane protein in the
reconstituted proteoliposomes. This enables us to study the lipid charge and thickness effects on
membrane protein stability. By using steric trapping method, we also investigated the unfolding kinetics
of bR under bilayer-like conditions. Surprisingly, unfolding of bR is extremely slow and the half-life for
this unfolding is on the order of several hours. Another large membrane protein, diacylglycerol kinase,
also shows really slow unfolding kinetics by this method. The high kinetic barrier to unfolding has
potential implications for both in vitro and in vivo folding of membrane proteins.
POST 06-364
Effects of Mercury Ion on the Structure and Function of E. coli Aquaporin Z
Qingsong Lin1, 2, Hu Zhou1, Lili Wang2
1
Department of Biological Sciences, National University of Singapore, Singapore, Singapore, 2NUS
Environmental Research Institute (NERI), National University of Singapore, Singapore
2+
Aquaporins (AQPs) are a family of membrane proteins that function as water channels. Mercury ion (Hg )
2+
was found to inhibit the function of most AQPs. Hg was reported to bind to Cys189 of human AQP1 at
the constriction region, which blocks the water channel. For the AQPs without cysteine residues at the
constriction region, such as the E. coli aquaporin Z (AQPz), the mechanism of mercury inhibitory effect
remains unclear. In the present study, recombinant AQPz was incorporated into proteoliposomes and the
mercury inhibitory effect was investigated with stopped-flow light scattering spectroscopy. The AQPz
2+
secondary structure changes in the presence of Hg were monitored with circular dichroism (CD)
2+
spectroscopy. The results suggested that the binding of Hg to AQPz disrupted the coiled coil
conformation of helixes. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) detected
233
POSTER ABSTRACTS
increased solvent accessibility of Phe10 of AQPz, corresponding to the CD results. HDX-MS also detected
dramatic decrease of solvent accessibility of one of the NPA motifs, suggesting blockage of the water
2+
channel, probably due to Hg -induced conformational changes. Site-directed mutagenesis studies
2+
revealed that Cys20 of AQPz was targeted by Hg to destabilize its tetrameric structure. This research
project is supported by a grant of the Singapore National Research Foundation under its Environmental &
Water Technologies Strategic Research Programme and administered by the Environment & Water
Industry Programme Office (EWI) of the PUB.
POST 06-365
Characterization Of The Calcium And Membrane Binding Properties Of The Hearing Related Protein
Otoferlin
Colin P. Johnson, Murugesh Padmanarayana, Nicole Hams, Ryan Mehl
Oregon State University, Corvallis, Oregon, US
Otoferlin is a transmembrane protein consisting of six C2-domains, proposed to act as a calcium sensor
for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of
the calcium binding domains is controversial. Further, it is currently unclear as to whether the calcium
binding affinity of otoferlin quantitatively matches the 30-100 uM intracellular pre-synaptic calcium
concentrations known to elicit exocytosis. To characterize the calcium and lipid binding properties of
otoferlin, we used isothermal titration calorimetry, liposome sedimentation assays and fluorescence
spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of
otoferlin bind multiple calcium ions with moderate (Kd= 25-95 mM) and low affinities (Kd=400-700 mM). It
was also determined that calcium enhanced liposome binding for domains C2B, C2C, C2D and C2E
whereas the C2F domain bound liposomes in a calcium independent manner. Further, domains C2C and
C2F, but not C2A, C2B, C2D or C2E bound PI(4,5)P2, and preferentially steer towards liposomes harboring
PI(4,5)P2. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural
amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid
bilayer of negatively charged liposomes in a calcium independent manner. Based upon these results, we
propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may
serve to target otoferlin to the plasma membrane in a calcium independent manner. This positioning
would facilitate fast calcium dependent exocytosis at the hair cell synapse.
POST 06-366
Towards a Crystal Structure Of The Hiv-1 Membrane Protein, Vpu
Arpan Deb2, 1, 3, William Johnson2, 1, 3, Dustin Srinivas2, 1, 3, Liqing Chen2, 4, Petra Fromme2, 4, Tsafrir LeketMor2, 1, 3
1
School of Life Sciences, Arizona State University, Tempe, Arizona, US, 2Center for Membrane Proteins in
Infectious Diseases, Arizona State University, Tempe, Arizona, US, 3Center for Infectious Diseases and
Vaccinology, The Biodesign institute, Arizona State University, Tempe, Arizona, US, 4Department of
Chemistry and Biochemistry, Arizona State University, Tempe, Arizona, US
Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency
Virus-1(HIV-1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4
receptors in T cells and also in the budding of virions. But there remain key structure/ function questions
regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe
our efforts to express VPU in bacteria, its purification and characterization. We report successful
expression of Vpu in the Escherichia coli expression system using the leader peptide pectate lyase B
234
POSTER ABSTRACTS
of Erwinia carotovora and also in fusion with Bacillus subtilis protein Mistic and the E. coli Maltose binding
protein. We also report successful detergent extraction, immobilized metal affinity-chromatography
purification and also further purification by size exclusion chromatography. The protein was biophysically
characterized using mass spectrometry, circular dichroism and dynamic light scattering experiments. We
also present preliminary data of our efforts to crystallize Vpu.
POST 06-367
Conformational Landscape Governing the Constitutive Activity of GPCRs
Ravinder Abrol1, 2, Caitlin E. Scott2, William A. Goddard III2, Kwang H. Ahn3, Debra E. Kendall3
1
Medicine, Cedars-Sinai Medical Center, Los Angeles, California, US, 2Chemistry, California Institute of
Technology, Pasadena, California, US, 3Pharmaceutical Sciences, University of Connecticut, Storrs,
Connecticut, US
The pleiotropy of G protein-coupled receptors (GPCRs) is enabled by their conformational flexibility
during activation. Many GPCRs exhibit constitutive activity, whose structural basis is not understood.
This study explored the conformational changes that underlie the constitutive activity of the CB1
cannabinoid GPCR by designing constitutively active mutants starting from an inactive CB1 mutant and
by designing inactive mutants starting from an active mutant. Ligand binding and GTPgS assay data (for
G protein coupling) had suggested that T210A mutant of the CB1 receptor was inactive and two mutants
T210I and L207A were more active than the wild-type (WT) receptor. Structure prediction of the WT and
mutant forms resulted in conformational changes consistent with known changes during GPCR
activation. It identified a unique salt-bridge interaction in the CB1 inactive mutant T210A between an
Arg residue on transmembrane helix 2 (TM2) and a conserved Asp residue on TM helix 6 (TM6), which
was proposed to keep the receptor fully inactive [Scott et al. (2013). Protein Science 22:101]. The WT
and other active receptor mutants lacked this interaction. To test this hypothesis, this salt-bridge
interaction was disrupted by designing two constitutively active double-mutants lacking the TM2+TM6
ionic lock, starting from the inactive T210A mutant. The GTPgS assay data confirmed that both these
mutants were constitutively active. The active L207A mutant that lacked the TM2+TM6 ionic lock was
then used to design an inactive double-mutant receptor that possessed that TM2+TM6 ionic lock. The
inactivity of this double mutant was confirmed by GTPgS assays, and then reversed by adding a third
mutation to rescue some of the constitutive activity [Ahn et al. (2013) Proteins 81:1304]. These data
strongly support the role of TM2+TM6 salt-bridge interaction in keeping the receptor inactive and also
show that the constitutive activity of this receptor is controlled by distinct changes in salt-bridge
interactions in the TM region. These changes underlying constitutive activity provide a “conformational
landscape” that can be modulated by extracellular stimuli like hormones to provide a more complete
structural picture of GPCR activation.
POST 06-368
Cell-free Translation Systems For Biophysical And Biochemical Characterization Of Proteins And
Protein Complexes
Feliza A. Bourguet1, Craig D. Blanchette1, Nicholas O. Fischer1, Paul J. Jackson1, Masood Z. Hadi2, Wei
He3, Brian K. Kay4, Kit S. Lam3, Ted A. Laurence5, Zachary Rogers3, John C. Voss6, Matthew A. Coleman1, 7
1
Biology and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, California,
US, 2NASA Ames Research Center, Mountain View, California, US, 3Center for Biophotonics, University of
California Davis, Sacramento, California, US, 4Biological Sciences, University of Illinois Chicago, Chicago,
Illinois, US, 5Condensed Matter and Materials Division, Lawrence Livermore National Laboratory,
Livermore, California, US,6Biochemistry and Molecular Medicine, University of California Davis,
235
POSTER ABSTRACTS
Sacramento, California, US, 7Radiation Oncology, University of California Davis, Sacramento, California,
US
Cell-free systems have been utilized to produce difficult to express proteins, such as toxic and membrane
bound proteins. Here we discuss how our laboratory has incorporated cell-free technologies to produce
labeled proteins, protein complexes and trans-membrane proteins in yields that are more than sufficient
for biophysical and biochemical characterization. Because cell-free translation is an open process, we
have adapted commercial systems for multiple high-throughput screening techniques dependent on the
suspected proteins function. For example, labels and co-factors can easily be added to synthesis reactions,
Lys
such as BODIPY-labeled tRNA , fluorescent protein tags, peptides, SNAP-tags, small molecule co-factors,
nanolipoproteins, lipids and fluorescently-labeled lipids. We also demonstrate that co-translation of
multiple proteins is achievable and this is one of the most efficient processes for solubilizing membrane
bound proteins. We have also shown that protein complexes of up to three polypeptides can be cotranslated within a single reaction. A wide range of active enzymes and receptors such as rhodopisins, Gprotein coupled receptors (GPCRs), kinases, cytokines, antibodies, proteases and cell wall hydrolases,
secretion system complexes have been studied. After purifying, proteins and protein complexes were
used for biophysical and biochemical characterization using fluorescence correlation spectroscopy (FCS),
circular dichroism (CD), electron microscopy (EM) and kinetic analysis. Overall, cell-free represents a
unique solution to address multiple bottlenecks in the production, purification and characterization of
proteins that in the past have been difficult to previously obtain.
POST 06-369
Membrane Proteins Can Have High Kinetic Stability
Robert Jefferson1, Tracy Blois2, 1, James Bowie1
1
UCLA, Los Angeles, California, US, 2Amgen, Thousand Oaks, California, US
Approximately 10% of water soluble proteins are considered kinetically stable with unfolding half-lives in
the range of weeks to millenia. These proteins only rarely sample the unfolded state and may never unfold
on their respective biological timescales. It is still not known whether membrane proteins can be
kinetically stable, however. Here we examine the subunit dissociation rate of the trimeric membrane
enzyme diacylglycerol kinase from Escherichia coli as a proxy for complete unfolding. We find that
dissociation occurs with a half-life of at least several weeks, demonstrating that membrane proteins can
remain locked in a folded state for long periods of time. These results reveal that evolution can use kinetic
stability to regulate the biological function of membrane proteins, as it can for soluble
proteins. Moreover, it appears that the generation of kinetic stability could be a viable target for
membrane protein engineering efforts.
POST 06-370
Advancing Membrane Protein Crystallography Using the LCLS
Mark Hunter2, Brent W. Segelke2, Nadia Zatsepin 1, Matt Coleman2, W. Henry Benner2, Stefan HauRiege2, Ching-Ju Tsai3, Xiao-dan Li3, Bill Pedrini3, Gebhard Schertler3, Matthias Frank2
1
Arizona State University, Tempe, Arizona, US, 2LLNL, Livermore, California, US, 3Paul Scherrer Institute,
Villigen, Villigen, Switzerland
X-ray crystallography has been the workhorse of structural biology, providing the majority of high
resolution protein structures. However, several challenges remain in x-ray crystallography, such as the
need to produce large, well ordered protein crystals and avoiding x-ray induced radiation damage, two
issues that are particularly acute for membrane protein structure determination. The advent of the LCLS
and other hard x-ray free electron lasers offers a possible remedy to these challenges thanks to the high
236
POSTER ABSTRACTS
peak brightness and short pulse duration of the source. The first crystallography experiments at LCLS
were carried out on the photosystem I complex, an over 1MDa integral membrane protein with 36 protein
subunits, from which electron density maps were obtained at ~8.5 Å resolution using 2keV x rays. More
recently, diffraction data of photosystem I were recorded to ~3 Å resolution using the LCLS, which were
the first diffraction patterns of a membrane protein crystal recorded to better than molecular resolution
using the LCLS. However, two-dimensional crystals (2D) of membrane proteins may offer a more natural
environment to study the structure and function of membrane proteins. As such, two-dimensional
crystallography of membrane proteins at the LCLS was also pursued, with diffraction data recorded to ~4
Å resolution from bacteriorhopsin, a membrane protein standard. Ultimately, the LCLS not only offers the
ability to outrun radiation damage but also offers the ability to probe ultrafast dynamics of membrane
proteins. Time-resolved 2D crystallography experiments of the early time points in the photocycle of
bacteriorhodopsin were performed, and preliminary data are currently being evaluated at ~4 Å
resolution. Although far from complete, the current results of membrane protein crystallography at LCLS
suggest great utility for the light source in furthering knowledge of structure and function of membrane
proteins.
Poster Session: Translation & Folding
POST 07-371
Elucidating the Structure And Dynamics Of Small Heat Shock Protein Complexes Using A Hybrid
Approach
Michelle Heirbaut1, Steven Beelen1, Esther Martin2, Frederik Lermyte2, Tim Verschueren2, Frank Sobott2,
Sergei Strelkov1, Stephen Weeks1
1
Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Brabant, Belgium, 2Chemistry,
University of Antwerp, Antwerpen, Belgium
Small heat shock proteins (sHSPs) are a highly conserved class of molecular chaperones that are part of
the cellular proteostasis network. Acting as holdases, sHSPs bind partially unfolded proteins without the
requirement for ATP. Their importance is underlined by the identification of congenital mutations in a
number of human homologues that are linked to diseases such as cataract. However, despite their long
recognized role in protein quality control, it remains unclear how the activity of sHSPs is modulated at the
sequence and structural level. The principle challenge in studying sHSPs is that they form large oligomeric
assemblies. These structures are based on dimeric building blocks that associate to form oligomers that
are polydisperse both in size and shape, and where the component subunits show a high turnover. This
complexity is further compounded in higher eukaryotes by the formation of hetero-oligomeric
assemblies composed of two or more homologues. Humans encode ten sHSPs (HSPB1-10) of which some
have have been shown to interact with each other. We are currently investigating the structure and
function of the complexes formed between HSPB1 and HSPB6, two sHSPs that are highly expressed in
smooth muscle. The complexes formed between them are highly polydisperse, ranging from 100 to 500
kDa. In order to elucidate the dynamics and structure of these entities we have employed a hybrid
structural approach. Using native mass spectrometry with isotopic labeling, we have characterized subunit
turnover and determined the size of the complexes formed. Curiously while both sHSPs in isolation show
stochastic subunit exchange at the monomer level, when mixed they form oligomers assembled from a
hetero-dimer, suggesting a sequence-based bias that preferentially stabilizes these mixed entities. By
237
POSTER ABSTRACTS
employing mutagenesis and SEC-coupled SAXS we have identified that a highly conserved sequence in
the unstructured N-terminal domain (NTD), that we show is a negative regulator of HSPB6 chaperone
activity, is essential for this preferential association. In addition, different regions of the NTD seem to
regulate the overall size and shape of the resultant oligomers. These findings provide initial insights into
the properties of human sHSP hetero-oligomers, illuminating the roles of particular sequences in dictating
the assembly and activity of these physiologically relevant species.
POST 07-372
Achieving Folding Cooperativity In A Physiological Environment
Nathan Gardner, Chiwook Park
Purdue University, West Lafayette, Indiana, US
During folding or unfolding, multimeric proteins often populate intermediate states that could aggregate
or cause a buildup of inactive protein. These situations can be avoided by folding cooperatively, only
populating the native and unfolded states. Here we investigate how the chemical environment can
regulate the folding cooperativity of a multimeric protein through urea-induced equilibrium unfolding.
From our previous stability-based E. coli proteome screen for ATP-binding proteins, we learned that ATP
significantly stabilizes cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein
whose enzymatic function does not require ATP. Interestingly, we found that dPGM unfolds in two
transitions with a monomeric intermediate in the absence of ATP, but the protein unfolds with a
cooperative, single transition in the presence of ATP. To elucidate the effect of ATP, we examined the
influence of adenosine, AMP, ADP, and GTP on dPGM folding. The stability of dPGM is directly correlated
with the number of phosphates per metabolite and is not influenced by the base. We also observed that
dPGM is stabilized by a wide range of salts, primarily by anions. Further, pyrophosphate stabilizes dPGM
1,000 fold better than phosphate. To test if ATP selectively stabilizes native dPGM by binding to the
positively charged active site, we constructed five dPGM mutants containing a different point mutation,
either arginine or lysine to alanine, within the active site. Four of the five mutants were significantly less
stabilized by ATP when compared to wild type, suggesting that ATP indeed binds to the active site of the
enzyme. Our result demonstrates how the chemical environment can stabilize dPGM by reducing
unfavorable Coulombic interactions in the active site, and that physiological concentrations of anions and
nucleotides can selectively stabilize native dPGM such that folding is cooperative.
POST 07-373
n→π* Interactions in Protein Structure and Folding
Robert W. Newberry, Ronald T. Raines
University of Wisconsin-Madison, Madison, Wisconsin, US
The folding of proteins is directed by a variety of noncovalent interactions, but current limitations in
protein structure prediction and protein design demonstrate that our understanding of these interactions
is incomplete. We show that amide carbonyl groups can engage in attractive interactions
through n→π* electronic delocalization, wherein a carbonyl oxygen donates electron density into an
empty antibonding orbital of another nearby carbonyl group. While the energy of these interactions is
modest, their abundance can make an immense contribution to protein stability. Moreover, these
interactions impart distinctive signatures to the structure and electronics of proteins. Finally, we present
evidence that these n→π* interactions can direct the folding of a polymer in the absence of hydrogen
bonding.
238
POSTER ABSTRACTS
POST 07-374
Structural And Dynamic Insights About Unfolding Intermediates In Four Amyloidogenic
Immunoglobulin Light Chains
Gilberto Valdes-Garcia, Cesar Millan-Pacheco, Nina Pastor
Facultad de Ciencias, Universidad Autonoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
Light chain amyloidosis (AL) is a misfolding disease characterized by the extracellular deposition of
immunoglobulin light chains (LCs) as insoluble aggregates [1]. Among the LCs families, lambda 6a is
highly frequent in AL patients. Its germline protein (6aJL2) and point mutants (R24G, P7S and D52A) are
good models to study fibrilllogenesis, because these mutations have drastic effects in stability and fibril
formation [2,3]. As yet, the conformational changes resulting in LC intermediates able to form fibrils have
not been characterized at the atomic level. Given that the native state of these proteins does not
aggregate, we perform molecular dynamics simulations at high temperatures (398, 448 and 498K) to take
them out of their native basin. This method allows us to sample the conformational landscape, to find
intermediates able to form fibrils. We found that the four proteins share a native-like intermediate
characterized by bending out the loop formed by the C'-C'' strands, in a hinge-like movement. This leaves
one edge of the beta sandwich unprotected, and eliminates an antiaggregation mechanism already
described for LCs [4]. Mutation R24G compromises the stiffness of the CDR1, that shrinks weakening the
connection of the CDR1 with the protective C'-C'' loop. Mutant D52A disrupts a negative patch, lowering
fluctuations in its vicinity, and P7S shows an increased resistance to lose secondary structure upon
denaturation. Despite the apparent stabilization, both D52A and P7S are less stable than 6aJL2,
suggesting a different unfolding pathway than that used by 6aJL2. Unfolding trajectories show that
mutant proteins are more prone to populate states with partially folded nuclei involving strands C, F and
B; these strands contain highly amiloidogenic sequences [5]. We propose that 6aJL2 and its mutants
populate different fibrillogenic intermediates, leading to variable efficiency to form amiloids.
[1]
Dispenzieri A et al (2012) Blood Rev. 26: 137; [2] del Pozo-Yauner L et al (2008) Proteins 72: 684; [3]
Hernández-Santoyo A et al (2010) J. Mol. Biol. 396: 280; [4] Richardson JS and Richardson DC (2002) Proc.
Natl. Acad. Sci, USA 99:2754; [5] Goldschmidt L et al (2010) Proc. Natl. Acad. Sci, USA 107:3487
POST 07-375
Mechanistic Insights Into The Folding Of The Trefoil-Knotted Proteins
Nicole C. Lim, Sophie E. Jackson
Chemistry, University of Cambridge, Cambridge, United Kingdom
The past decade has seen the emergence of a new class of proteins that possess an intriguing knotted
topological feature in their structures formed by the path of the polypeptide backbone (1, 2). Elucidating
when and how a polypeptide chain knots during the folding represents a significant challenge to the
protein folding field as the knotted topology imposes additional complexity to the folding landscape.
Most of the experimental investigations on knotted proteins have been focussed on two bacterial
trefoil-knotted α/β methyltransferases, YibK and YbeA (2-4). Recently, with the use of a cell-free
expression system and pulse-proteolysis kinetic experiments, Mallam and Jackson were able to
investigate the folding rates of nascent chains of knotted proteins after they were first synthesised by
the ribosome (4). By using the same cell-free expression system and pulse-proteolysis kinetic
experiments, this study investigates the mechanism of knotting by monitoring the effects of an
additional protein domain on the folding rates of YibK and YbeA when it is fused to either the amino
terminus, carboxy terminus or to both termini. Here, we demonstrate that the fusion of the additional
protein domain to either the carboxy terminus or both termini of the knotted proteins retards the rate
of folding, indicating that the threading motion is hindered. This suggests that the C-terminus is critical
239
POSTER ABSTRACTS
in the threading of the polypeptide chain to form a knot and thus provides the first experimental
evidence of the knotting mechanism. In addition, we also investigate the effect of the GroEL-GroES
chaperonin on the folding of these fusion proteins. Our results shed light on the role of molecular
chaperones on the folding of knotted proteins, thus giving us more insights as to how knotted proteins
have withstood evolutionary pressures despite their complex topologies and intrinsically slow rates of
folding. 1. Boelinger D, Sulkowska JI, Hsu H-P, Mirny LA, Kardar M, et al. 2010. Plos Computational
Biology 6 2. Virnau P, Mallam A, Jackson S. 2011. Journal of Physics: Condensed Matter 23 3. Mallam
AL. 2009. Febs Journal 276: 365-75 4. Mallam AL, Jackson SE. 2012. Nature Chemical Biology 8: 147-53
POST 07-376
Intermolecular Interactions in a Blood Clotting Mechanism Led to Protein Folding Theory
Harold A. Scheraga
Chemistry and Chemical Biology, Cornell University, Ithaca, New York, US
Our experimental determination of the mechanism of the thrombin-fibrinogen interaction led to
formulation of theory for formation of side-chain hydrogen-bonding and hydrophobic interactions.
Resulting experimental locations of such interactions in RNase A led to distance constraints for ultimate
formulation of ECEPP, an all-atom potential energy force field. ECEPP was used to compute the 3D
structure of the fibrous protein collagen and the globular 46-residue Protein A. To treat larger proteins, a
UNited RESidue coarse-grained model (UNRES) was developed, with applications in several CASP proteinstructure-prediction exercises. An effective homology-modeling algorithm was developed for use
together with UNRES to enhance its efficiency. In addition to protein-structure prediction, UNRES has
been applied to dynamics and thermodynamics of protein folding.
POST 07-377
Molecular Mechanism Of Nuclear Transport Mediated By Flexible Amphiphilic Proteins
Shigehiro Yoshimura, Msahiro Kumeta, Kunio Takeyasu
Kyoto University, Kyoto, Japan
Karyopherin β family proteins mediate the nuclear/cytoplasmic transport of proteins larger than the size
barrier of the nuclear pore complex (NPC), although they are substantially larger than the minimal NPC
size limit. To elucidate the molecular mechanism underlying this paradoxical function, we focused on their
unique structures called HEAT repeats, which consist of repetitive amphiphilic α-helices, and performed
intensive structural and functional analyses. We found that not only karyopherin β family proteins but also
other proteins with HEAT repeats could pass through the NPC by themselves, and serve as transport
mediators for their binding partners. Spectroscopic analyses and molecular dynamics simulation revealed
that they undergo reversible conformational changes in tertiary structures, but not in secondary
structures, depending on the presence of hydrophobic groups. In vitro crosslinking of karyopherin β
protein resulted in the reduction of influx rate across the nuclear envelope in in vitro transport assay,
indicating that conformational flexibility is necessary for their nuclear translocation. These
results demonstrate a novel function for amphiphilic proteins as potential transport receptors.
240
POSTER ABSTRACTS
POST 07-378
Native State Dynamics Of The Prion Protein Probed By Hydrogen Exchange And Mass
Spectrometry
Roumita Moulick, Jayant B. Udgaonkar
Biophysics and Biochemistry, National Centre For Biological Sciences, Bangalore, Karnataka, India
C
The prion protein (PrP ) appears to be unusually susceptible to conformational changes, and unlike nearly
Sc
all other proteins, it can be converted to alternative misfolded conformations (PrP ), which is the key
C
event in prion pathogenesis. The easy convertibility of PrP is indicative of a conformationally flexible
native form and in fact, the conformational flexibility of the native form has been reported to be critical in
prion conversion. A quantitative understanding of the basis of this structural plasticity in terms of stability
of the protein by a detailed thermodynamic characterization of unfolding of the mouse prion protein
indicated high levels of structural fluctuations in the native state (N) of the protein as indicated by an
unusually high native state heat capacity. In the present study, a structural and energetic characterization
of the native state of the wild type, full length, mouse prion protein (MoPrP 23-231) has been done in
solvent conditions where it has a high propensity to convert into aggregated forms, using hydrogen
exchange in conjunction with proteolytic fragmentation and mass spectrometry: a probe sensitive to the
presence of minimally populated conformations. The native state of the prion protein is observed to be at
equilibrium with multiple, sparsely populated, non-native conformers or partially unfolded forms, which
remain otherwise undetected by global probes. It seems possible that these sparsely populated nonnative
conformations (N* states) may act as monomeric precursors to misfolded oligomeric forms, thereby
initiating the conversion of monomer to multiple misfolded conformations, as has been identified for few
other proteins.
POST 07-379
Mutations in the Bacterial Ribosomal Protein S12 Influence Aminoglycoside Antibiotic And
Ribosome Dynamics
Joanna Panecka3, 1, Cameron Mura4, Joanna Trylska2
1
Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw,
Warsaw, Poland, 2Centre of New Technologies, University of Warsaw, Warsaw, Poland, 3Department of
Biophysics, University of Warsaw, Warsaw, Poland, 4Department of Chemistry, University of Virginia,
Charlottesville, Virginia, US
The ribosomal protein S12 that neighbors the aminoacylated-tRNA binding site (A-site) in the small
ribosomal subunit contributes to the fidelity of decoding. The S12 sequence with about 120 amino acids
is highly conserved in bacteria. The globular domain of S12 is located near the interface with the large
subunit and the S12 tail is anchored inside the small subunit. Experiments show that certain mutations
in the S12 protein can inactivate paromomycin, an aminoglycosidic antibiotic, even when this antibiotic
is already bound to its target - A-site in the small subunit. This inactivation mechanism is likely of
dynamical origin. Therefore, we have examined how the S12 mutations affect the conformational
dynamics of the region of the ribosome involving S12 and aminoglycoside binding site. We conducted
all-atom molecular dynamics simulations in explicit solvent of a ribosome fragment that includes the S12
protein, the A-site RNA and its surroundings within 30A. Our simulations, with an aggregate length of 0.9
microseconds, were performed with the wild-type S12 protein and its K42A and R53A 'hyper-accurate'
variants either with or without paromomycin. The aim was to analyze the link between S12 mutations
and the antibacterial action of this aminoglycoside. From molecular dynamics trajectories we observed
that the binding mode of paromomycin in the A-site did not change in any of the S12 mutants, in accord
with experimental data. However, the A-site mobility was affected by stacking interactions between
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POSTER ABSTRACTS
A1493 (of helix 44 in the small subunit) and A1913 (of Helix 69 in the large subunit), and by contacts
between A1492 and a flexible side-chain K43 of the S12 protein. In the latter case, the antibiotic bound
in the A-site reduced the frequency of hydrogen bonds between K43 and A1492, which may be a
contributing factor to the miscoding effect of the antibiotic. On the contrary, these K43···A1492
interactions in the S12 mutant systems with paromomycin were unchanged, consistent with the known
resistance of hyper-accurate mutants to aminoglycoside-induced miscoding. We also found that binding
of paromomycin, or the existence of the S12 mutations, increased stacking between A1493 and A1913.
Such stacking interactions were not found in the wild-type S12 system without paromomycin. Our work
suggests possible links between the biological effects of ‘hyper-accurate’ mutations in the S12 protein
and conformational properties of the ribosome.
POST 07-380
Curious Characteristics Of A Mutant Chaperonin GroeL With Multiple Cysteines In The Central
Cavity
Tomohiro Mizobata, Shuhei Fusa, Masashi Ikeda, Kunihiro Hongo, Yasushi Kawata
Biotechnology, Tottori University Graduate School of Engineering, Tottori, Tottori Prefecture, Japan
The bacterial chaperonin GroEL facilitates the refolding of various denatured proteins by binding to
aggregation-prone substrate protein molecules and sequestering them within its characteristic central
cavity. This highly dynamic process involves multiple interactions between GroEL, the co-chaperonin
GroES, and the refolding protein. The molecular cycle is orchestrated by the binding and hydrolysis of
ATP, by GroEL. We were interested in finding out if certain proteins with native disulfide bonds could
interact with the inner wall of the GroEL central cavity through disulfide exchange while encapsulated. To
this end, we introduced two cysteine residues that compose a putative "disulfide exchange (CXXC) motif"
278
281
into a modified (native cysteine-depleted) version of E. coli GroEL. The site ( Cys-Pro-Gly-Cys ) was
chosen so that the introduced cysteines would protrude into the central cavity of GroEL in the ATP-bound
open conformation (See image). The mutant chaperonin (GroEL C-All-A CPGC) was expressed robustly
in E. coli cells; however, during purification it was found that the mutant GroEL bound the wild type cochaperonin GroES extremely tightly, and GroES could not be removed without denaturing the
sample. Accordingly, the purified sample failed to display any detectable ATPase activity. Curiously, this
purified binary complex could facilitate the refolding of multiple proteins such as rhodanese and malate
dehydrogenase, in efficiencies comparable to the wild type. This interesting result was probed in more
detail, and further experiments revealed that: 1. The mutant GroEL could be expressed independently
from GroES; such samples retained protein refolding ability and displayed a very weak ATPase activity. 2.
Observation using electron microscopy failed to reveal any obvious differences in the GroEL:GroES binary
complexes formed by the mutant. Based upon our present findings, we suspect that GroEL C-All-A CPGC
might be facilitating the refolding of rhodanese and malate dehydrogenase through a novel mechanism
that does not require ATP hydrolysis. We would like to propose in our presentation some plausible
mechanisms by which these peculiar results may be explained, as well as additional data to support our
ideas.
POST 07-381
Untangling Ribosome Biogenesis Using Quantitative Mass Spectrometry, Electron Microscopy And
Chemical Probing
Joseph H. Davis1, Nikhil Jain2, Admad Jomaa3, Joaquin Ortega3, Robert Britton2, James R. Williamson1
1
Integrative Computational and Structural Biology, La Jolla, California, US, 2Microbiology & Molecular
Genetics, Michigan State University, East Lansing, Michigan, US, 3Biochemistry and Biomedical Sciences,
McMaster University, Hamilton, Ontario, Canada
242
POSTER ABSTRACTS
Ribosome biogenesis is an essential and complex cellular process involving the synthesis, folding,
chemical modification, and assembly of 3 large RNAs and 55 proteins. This process is accomplished
efficiently with cells capable of synthesizing more than 100,000 ribosomes/hour. An array of largely
uncharacterized co-factors are essential in guiding subunit assembly by helping to ensure that the
maturing particles avoid kinetics traps. Although early pioneering studies provided maps for the
thermodynamic interdependencies of r-protein binding in vitro, we still lack a detailed mechanistic view of
how ribosomes are assembled so efficiently in vivo. Here, we employ quantitative mass spectrometry,
chemical probing and cryo-EM to characterize the structure and protein composition of a variety of
assembly intermediates. To enrich for these particles, we generated a series of bacterial strains that allow
for the specific cellular depletion of essential assembly co-factors (e.g. B. subtilis RbgA) or ribosomal
proteins (e.g. E. coli S2 and L17). These experiments reveal proteins that are specifically depleted from
various assembly intermediates and uncover detailed interdependencies in r-protein binding and subunit
maturation in vivo. Comparison of protein occupancy between these depletion strains allows for the denovo construction of a subunit assembly map and suggests a highly parallel assembly pathway in vivo.
Clustering analysis of these datasets identified groups of proteins with tightly correlated binding patterns
implying cooperatively in their binding. Additionally, we characterized these particles structurally using
cryo-EM and chemical probing. By mapping the protein binding patterns onto these structures, we
identified structural domains of the ribosome that undergo concerted assembly and maturation. We
have complemented this work with a novel MS-based pulse labeling approach that can distinguish
physiologically-relevant assembly intermediates from dead-end particles. Further, for each ribosomal
protein, this approach provides an estimate of the time required from protein synthesis to incorporation
into a complete 70S particle; information that can be used to determine the in vivo kinetics of complex
assembly. The mass spectrometry and structural techniques we’ve developed are easily generalized and
should prove useful in the study of a variety of macromolecular assembly problems.
POST 07-382
Study of E.coli GroEL Using Stopped-Flow Analysis And Circular Permutation
Toshifumi Mizuta1, Tatsuya Uemura2, Kunihiro Hongo1, 2, Yasushi Kawata1, 2, Tomohiro Mizobata1, 2
1
Department of Chemistry and Biotechnology, Tottori University, Tottori, Japan, 2Department of
Biomedical Science, Tottori University, tottori, Japan
Escherichia coli GroEL is a member of the chaperonins, a well-conserved protein folding assistant
necessary for cell growth. The GroEL subunit consists of three different functional domains. The apical
domain contains the GroES and substrate protein binding sites, the equatorial domain contains the
ATPase binding site which controls the overall functional cycle, and these domains are linked by the
intermediate domain. In order to monitor the dynamic changes of the apical domain, we used stoppedflow analysis introduced a fluorescent tryptophan residue mutant (GroEL R231W). Previous studies have
shown that GroEL undergoes five kinetically distinguishable transitions (Phases A to D, Phase S) during
the process of encapsulation. However, the role of these five phases have not been completely
understood. To probe the five phases in more detail, we introduced a fluorescent tryptophan residue to
monitor the dynamic movements of the apical domain of circularly permuted GroEL (GroEL CP86
R231W, GroEL CP376 R231W), single ring GroEL (GroEL SR1 R231W), and ATPase-impaired GroEL (GroEL
D398A R231W). Circular permutation involves modifying the amino acid sequence of the target protein
so that N- and C-terminal amino acids are shifted to a different portion of the protein molecule. We also
inserted cysteine residues to GroEL CP86 in the vicinity of the N- and C- terminal (GroEL CP86 C4). This
mutant was designed to link the disconnected N- and C- termini by disulfide bond under oxidative
conditions. From the results of stopped-flow analysis of GroEL CP86 R231W, we could not detect apical
domain conformational changes. In spite of this, this mutant was able to assist the folding of MDH
efficiently. This result, suggested that apical domain movement were not strictly necessary for refolding
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POSTER ABSTRACTS
of some substrate proteins. Also, from stopped-flow analysis of GroEL SR-1 and GroEL D398A variants,
we observed reduced dissociation rate of denatured protein. These results suggested that the presence
of two GroEL rings and a specific conformational rearrangement in GroEL triggered by ATP binding
contribute significantly to the rapid release of substrate protein from the GroEL apical domain.
POST 07-383
A Global Machine Learning-Based Scoring Function For Protein Structure Prediction
Andrzej Kloczkowski1, 2, Eshel Faraggi2, 3
1
Pediatrics, The Ohio State University, Columbus, Ohio, US, 2Battelle Center for Mathematical Medicine,
Nationwide Children's Hospital, Columbus, Ohio, US, 3Indiana University, Indianapolis, Indiana, US
We present a knowledge-based function to score protein decoys based on their similarity to native
structure. A set of features is constructed to describe the structure and sequence of the entire protein
chain. Furthermore, a qualitative relationship is established between the calculated features and the
underlying electromagnetic interaction that dominates this scale. The features we use are associated with
residue–residue distances, residue–solvent distances, pairwise knowledge-based potentials and a fourbody potential. In addition, we introduce a new target to be predicted, the fitness score, which measures
the similarity of a model to the native structure. This new approach enables us to obtain information both
from decoys and from native structures. It is also devoid of previous problems associated with
knowledge-based potentials. These features were obtained for a large set of native and decoy structures
and a back-propagating neural network was trained to predict the fitness score. Overall this new scoring
potential proved to be superior to the knowledge-based scoring functions used as its inputs. In particular,
in the latest CASP (CASP10) experiment our method was ranked third for all targets, and second for freely
modeled hard targets among about 200 groups for the top model predictions. Ours was the only method
ranked in the top three for all targets and for hard targets. This shows that initial results from the novel
approach are able to capture details that were missed by a broad spectrum of protein structure prediction
approaches. Source codes and executable from this work are freely available at http://mathmed.org and
http://mamiris.com/.
POST 07-384
Effect of Mg 2+ in the Structure And Thermal Stability Of Enolase From Trichomonas vaginalis.
Elibeth Mirasol Meléndez1, Jorge L. Rosas Trigueros2, Luis G. Brieba de Castro3, Rossana Arroyo
Verástegui4, Claudia G. Benítez Cardoza1
1
Biochemistry, Instituto Politécnico Nacional, Mexico, Mexico City, Mexico, 2Escuela Superior de
Cómputo, Instituto Politécnico Nacional, Mexico, Mexico, Mexico, 3Structural Biochemistry, Centro de
Investigación y Estudios Avanzados LANGEBIO, Guanajuato, Irapuato, Mexico, 4Infectómica, Centro de
Investigación y Estudios Avanzados, Mexico, Mexico City, Mexico
Trichomonas vaginalis is the causing agent of the most common non-viral sexually transmitted disease
known as trichomoniasis. Enolase is an enzyme that catalyzes the reversible dehydration of 22+
phosphoglycerate to phosphoenolpyruvate, using Mg as cofactor. Enolase has also been described as a
plasminogen receptor within Trichomonas vaginalis and might be implicated in the host-parasite
virulence process. Similarly to other glycolytic enzymes, enolase is considered as an attractive target for
rational drug design. Even though, a thorough characterization of its conformational plasticity that might
be associated to its multifunctionality is still needed. Here we present a combination of in vitro and in
silico tools to explore the conformational space of apo and holo-enolase from T. vaginalis. Firstly, the
model of the three-dimensional structure of the enzyme was obtained by the I-Tasser software, which
combines threading and Ab initio modeling. Furthermore, we sampled different possible conformations of
enolase by Molecular Dynamics Simulation (MDS). In addition, we studied the effect of three different
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POSTER ABSTRACTS
2+
buffer conditions and the presence of Mg on the conformation and thermal stability of recombinant
enolase from T. vaginalis, by circular dichroism and intrinsic fluorescence spectroscopies. Commonly, it
has been observed that enolase from several biological species is stabilized by its cofactor. In the case of
2+
enolase from T. vaginalis we concluded that Mg might have stabilizing or destabilizing effects
depending on the cosolutes present the protein solution.
POST 07-385
Controlling Nanostructures Of Insulin Amyloid Fibrils Using Metal Ions
Misaki Yokoyama, Yoshito Furuie, Motonari Tsubaki, Hiroshi Hori, Takamasa Nishida, Kazuo Eda, Eri
Chatani
Chemistry, Kobe university, Kobe, Hyogo, Japan
Amyloid fibrils are generally considered aberrant protein aggregates associated with the pathology of
numerous human diseases, but their well-ordered nanostructures have a potential appeal as a new
material for functional purposes in the field of nanotechnology. To control physicochemical and
physicomechanical properties of amyloid fibrils, polymorphism, a property with which a variety of fibril
structures are formed even from one protein sequence, will be useful. In this study, we have attempted to
produce polymorphic fibrils of insulin by altering initial association state with zinc ions; insulin forms a
hexamer with zinc ions preferably thorough their coordination to histidine residues. As a result of
incubation of insulin under the agitating conditions at pH7.5 and 37°C in the presence or absence of zinc
ions, we could obtain insulin fibrils for both conditions. The amyloid fibrils formed in the presence of zinc
had higher stability against pH-induced dissociation than those formed in its absence, indicating that
metal ion is an effective factor to induce polymorphism of insulin fibrils. Interestingly, a different type of
fibrils was formed when insulin was incubated with copper ions, and it is suggested that more meticulous
control of fibril structures may be achieved by using different metal ions.
POST 07-386
Structural and Thermodynamic Characterization of the X−Dimer of Human P−Cadherin:
Implications for Homophilic Cell Adhesion
Shota Kudo1, Jose Caaveiro1, Satoru Nagatoishi1, Takao Hamakubo1, Tatsuhiko Kodama1, Tadashi
Matsuura2, Yukio Sudou2, Kouhei Tsumoto1
1
The University of Tokyo, Tokyo, Japan, 2Perseus Proteomics Inc., Tokyo, Japan
Cadherins are calcium−dependent cell adhesion proteins involved in selective cell−cell recognition and
the development and maintenance of solid tissues. Human P−cadherin is a member of classical cadherin
family weakly expressed in basal epithelia, but over−expressed in pancreatic, breast and lung cancers.
P−cadherin is therefore a promising target in the battle against cancer. However, unlike other cadherins,
little is known about the properties of P−cadherin at the molecular and cellular levels. In this report we
have characterized human P−cadherin from both structural and thermodynamics standpoints. We
demonstrate that human P−cadherin dimerizes in the orthodox strand−swap dimer (ss−dimer) as
reported for other members of this family of adhesive proteins. Importantly, human P−cadherin also
dimerizes in a second stable conformation known as the X−dimer, a key intermediate for ss−dimerization.
This is just the second example of X−dimerization in classical cadherins. However, it is still unclear how the
X−dimer contributes to cell sorting and segregation. We further characterized the X−dimer of human
P−cadherin by a combination of structural, thermodynamic and spectroscopic techniques. The formation
of the X−dimer is driven by a favorable change of enthalpy, suggesting the formation of specific
interactions at the molecular level (e.g. H−bonds) between the cadherin protomers, but not with other
cadherins. This idea is corroborated during the X−dimerization of P−cadherin in the presence of
245
POSTER ABSTRACTS
stoichiometric amounts of E−cadherin. Taken together, the results suggest that differences in molecular
interactions specific to each cadherin contribute to the homophilic dimerization of the stable ss−dimer.
We propose that the X−dimer is the first checkpoint in the early stages of cell−cell recognition mediated
by cadherins.
POST 07-387
A Novel Protein Fold within the N-terminus of a Streptococcal Adhesin Mediates Proper Folding,
Function, and Stability
Kyle P. Heim1, 2, Paula Crowley1, Shweta Kailasan2, Robert McKenna2, Jeannine Brady1
1
Oral Biology, University of Florida, Gainesville, Florida, US, 2Biochemistry and Molecular Biology,
University of Florida, Gainesville, Florida, US
Streptococcus mutans is a ubiquitous oral pathogen and a primary causative agent of dental
caries. Adhesin P1 (AgI/II) is localized on the surface of S. mutans and interacts with the host
glycoprotein complex salivary agglutinin. Recent crystal structures of P1 display an unusual structure in
which the 1561 amino acid protein folds back upon itself to form an elongated alpha-polyproline-II
hybrid helical stalk separating two independent adherence domains, one at the apex and one at the
base of the molecule. We have now discovered that proper function of P1 on the surface of S.
mutans requires an interaction involving the N- and C-terminal portions of the protein. In order to
characterize this interaction we employed several biophysical techniques including: isothermal titration
calorimetry, circular dichroism, surface plasmon resonance, differential scanning calorimetry and x-ray
crystallography. Utilizing recombinant N-terminal (a.a. 39-308) and C-terminal (a.a. 921-1486)
fragments, we have demonstrated the formation of a stable high-affinity complex between these
portions of the protein. Furthermore, the presence of the N-terminal fragment was found to contribute
to the folding and increase the functionality of the C-terminus. We have also shown that a 106 amino
acid segment within the N-terminal region of P1 contributes to the proper folding and function of the
full-length recombinant molecule and increases the stability of the protein’s elongated hybrid helical
stalk. Finally, the N-terminal/C-terminal protein complex has now been crystallized and diffraction data
collected out to 2.0Å. In our x-ray crystallography model the N-terminus appears to form a novel
protein fold that functions as a scaffold to bridge the N-terminal and C-terminal portions of the protein
while subsequently locking P1’s hybrid helical stalk into place. This x-ray crystallography data agrees
well with our previous biophysical data and now provides mechanistic insight into how the N-terminus
helps support folding, function and stability of the full length P1 molecule. This information will enable
interpretation of existing and future preventative therapies, and will also inform biological studies to
evaluate bacterial adhesion and biofilm formation.Funding: NIH Predoctoral Fellowship T90 DE02199003, University of Florida Alumni Fellowship, and National Institutes of Health Grants R01DE08007 and
R01DE21789 (NIDCR)
POST 07-388
Folding of Collagen Heterotrimeric Helices via Cation-π Interactions
Jia-Cherng Horng, Chu-Harn Chiang, Tang-Chun Kao
Chemistry, National Tsing Hua University, Hsinchu, Taiwan
Collagen is the most abundant protein in animals, and exists as the most predominant component of the
extracellular matrix. It is a right-handed triple helix composed of three parallel polyproline II helices.
Naturally occurring collagen triple helices are composed of all identical (AAA, homotrimer), two different
(AAB, heterotrimer), or three different (ABC, heterotrimer) polypeptide chains. The types of heterotrimeric
structures are of critical importance in understanding the extracellular matrix since many natural collagens
are of type AAB or ABC, and can better mimic most natural collagens. Herein, we prepared a series of
246
POSTER ABSTRACTS
collagen-related peptides (CRPs) in which cationic (arginine) and aromatic (phenylalanine) residues are
incorporated to explore the folding of collagen heterotrimeric helices via cation-π interactions. Circular
dichroism (CD), differential scanning calorimetry (DSC), and NMR measurements show that the CRPs can
fold into heterotrimeric helices via favorable cation-π interactions between polypeptide chains. By
controlling the mixing molar ratios of cationic and aromatic CRPs in solution, we could obtain the
heterotrimers consisted of different numbers of cationic and aromatic CRPs. We have demonstrated that
formation of the collagen heterotrimers can be induced by interchain cation-π interactions. The results
provide a new strategy to assemble CRPs into heterotrimeric structures.
POST 07-389
Loss of Conformational Entropy in Protein Folding Calculated using Realistic Ensembles (with
implications to NMR-based calculations)
Tobin R. Sosnick1, 4, Michael C. Baxa1, Esmael Haddadian2, Karl F. Freed3, 5
1
Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, US, 2Biological Sciences
Collegiate Division, University of Chicago, Chicago, Illinois, US, 3Chemistry, University of Chicago,
Chicago, Illinois, US, 4Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois,
US, 5James Franck Institute, University of Chicago, Chicago, Illinois, US
The determination of the loss of conformational entropy, a major term in the thermodynamics of protein
folding, has proven challenging. We determine this loss using explicit solvent simulations of both the
native protein and a realistic denatured state ensemble. For ubiquitin at T = 300 K, the conformational
-1
entropy loss per residue is TΔS=1.4 kcal·mol , with only 18% accounted due to the loss side chain
entropy. Our values are up to three-fold different than prior values because of the use of more accurate
ensembles and the correction for correlated motions. Buried side chains lose only a factor of ΩU/ΩN ~ 1.3
in the number of conformations available per rotamer. The entropy loss is different for helical and sheet
-1
residues, which lose a factor of 13 and 5 conformations, respectively (TΔShelix-sheet = 0.5 kcal·mol ). This
difference is due to the smaller motions of helical residues, a property not fully reflected in NH and C=O
NMR order parameters. Our results have implications for folding and binding thermodynamics, including
estimates of solvent ordering and microscopic entropies using NMR methods.
POST 07-390
Rare Example Of A Protein Where An Isolated Domain Is More Stable Than The Full-Length
Swati Bandi, Surinder Singh, Krishna Mallela
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of Colorado Anschutz Medical Campus, Aurora, Colorado, US
Studying the contribution of individual domains to protein structure and function is of considerable
interest over the years. Domains are in general less stable than the corresponding full-length proteins.
Here, we report an exceptional case of utrophin tandem calponin-homology (CH) domain. Isolated Cterminal CH domain (CH2) is both thermodynamically and kinetically more stable than the full-length
tandem CH domain. Reversible, equilibrium denaturant melts using both circular dichroism and protein
fluorescence signals show that the CH2 is thermodynamically more stable by 4.0 kcal/mol when compared
with the full-length tandem CH domain. Thermal melts indicate that CH2 unfolds at a higher temperature
(15°C) than the full-length protein. Stopped-flow kinetics indicate that the CH2 unfolds slower (by 3 times)
and folds faster (by 7 times) than the full-length protein, suggesting the higher kinetic stability of CH2.
Analytical ultracentrifugation, size-exclusion chromatography, and dynamic light scattering show that
both CH2 and the full-length protein are monomers in solution, confirming that the higher stability of
CH2 is not due to formation of oligomers. Thus, the utrophin tandem CH domain is a rare example in
which an isolated domain is more stable than the corresponding full-length protein.
247
POSTER ABSTRACTS
POST 07-391
Analysis of Several Monomeric Mutants of Triosephosphate Isomerase
Misrain E. Gurrola Acosta1, Maria E. Chánez2, Edgar Vazquez Contreras1
1
Natural Sciences, UAM-Cuajimalpa, Mexico, DF, Mexico, 2Vascular Cerebral Pathology, Instituto
Nacional de Neurología y Neurocirugía, México, DF, Mexico
In this work we present the results of the conformational and functional analysis of several monomeric
mutants of the Triosephosphate Isomerase (TIM) enzyme, making special emphasis in the Trypanosome
cruzi mutant (monoTcTIM), which has been designed, synthetized, purified and characterized by our
1
team . In the first hand, the PDB files of these proteins were obtained and the similarities and differences
in its three dimensional-structures were obtained. It was found that the proper TIM barrel scaffold of all
the molecules studied is conserved, despite the variations in their primary sequence because all they have
different original engineered mutations. Regarding the catalytic residues (see Figure 1), some differences
were found. For all the mutants the three-dimensional conformation of the catalytic residue GLU (167 or
16), is almost the same, however many differences were observed for LYS (13 or 14) and HYS (95-96).
These variations are maybe related with the drastic decrease in the catalytic parameters (Vmax, Km and
Kcat) when compared the mutants with the wild-type enzyme, because these residues are in direct contact
with the substrate during catalysis. In particular, for monoTcTIM, the side chain of the catalytic HIS residue
2
is not as distant from its wild type position as in the case of monoTbTIM , one monomeric mutant of TIM
from Trypanosoma brucei. This observation could be consistent with the differences in activity of the first,
regarding to the latter. The catalytic and three-dimensional differences between al the analyzed mutants
are reported, and their roll in the dimeric wild type nature of this enzyme discussed. 1.- Zárate-Pérez et
al. 2009; Biochem Biophys Res Comm. 382:626-630. 2.- Borchert et al. 1993; Prot Design. 91:15151518. This work was supported by CONACyT México (47310308)
POST 07-392
Probing the Denatured State of a Knotted Protein
David J. Burban1, Dominique Capraro1, Joanna Sulkowska2, Patricia Jennings1
1
Chemistry & Biochemistry, University of California, San Diego, San Diego, California, US, 2Chemistry,
University of Warsaw, Warsaw, Poland
Since their discovery, knotted proteins have become an area of exciting study in protein folding. Current
studies have yielded some important discoveries, including that chaperones are not needed in the
knotting of the structure and that knot formation happens late in the folding process (1-2). However, in
vitro studies have yet to elucidate if the knot can be completely removed in the denatured state. To probe
this question we have studied the SPOUT methyltransferase from Thermatoga Maritima (PDB code 1O6D),
which contains a 30 residue deep C-terminal trefoil knot in its folded state. Unfolding of 1O6D is complex
in that waiting well beyond five half-lives for equilibration, as judged from kinetic studies, results in shifts
in the unfolding and refolding “equilibrium” curves. This shift is uncoupled from simple unfolding and
may be an indicator of untying of the knot in the unfolded state. In order to further understand this shift
in the observed “equilibrium” transitions of 1O6D, a combination of in silico, experimental folding and
Nuclear Magnetic Resonance experiments will be discussed. 1) Sulkowska, J. I., Sulkowski, P., and Onuchic,
J. (2009) Proceedings of the National Academy of Sciences of the US of America 106, 3119-3124. 2)
Mallam, A. L., and Jackson, S. E. (2012) Nature Chemical Biology 8, 147-153.
POST 07-393
Transitions Between Different Side-Chain Conformations In Hydrophobic Residues
Diego Caballero1, 2, Corey S. O'Hern3, 1, 2, Lynne Regan4, 2
248
POSTER ABSTRACTS
1
Physics, Yale University, New Haven, Connecticut, US, 2Integrated Graduate Program in Physical and
Engineering Biology, Yale University, New Haven, Connecticut, US, 3Mechanical Engineering and
Materials Science, Yale University, New Haven, Connecticut, US, 4Molecular Biophysics & Biochemistry ,
Yale University, New Haven, Connecticut, US
We now have Angstrom-level resolution of the atomic positions for thousands of protein crystal
structures. From these structures, we can determine the probability distributions of the side-chain dihedral
angles for each type of amino acid. However, we lack a predictive understanding of these distributions,
for example, are the shapes of the distributions determined primarily by steric or electrostatic interactions
and are they dominated by local interactions within an amino acid or by longer-ranged
interactions? Similarly, we do not have a fundamental understanding of what interactions determine the
energy barriers that control transitions between amino acid side-chain conformations. To address these
questions, we performed all-atom molecular dynamics simulations of hydrophobic residues (Leu and Ile)
using a simple model of dipeptides that includes steric interactions plus stereochemical constraints. We
show that transitions between different side-chain conformations are strongly coupled with bond-angle
fluctuations formed by atoms within the side chain. However, transitions between side-chain
conformations obtained from the Amber and CHARMM force fields are not strongly correlated to changes
in the bond-angle distributions. These results emphasize the importance of steric interactions and
stereochemical constraints in determining side-chain conformational preferences for hydrophobic
residues.
POST 07-394
N-terminal Domain Of Luciferase Controls Misfolding Avoidance
Zackary N. Scholl, Weitao Yang, Piotr Marszalek
Duke University , Durham, North Carolina, US
The proteomes of all forms of life are predominated by large proteins. In contrast to small, one-domain
proteins, little is known about folding pathways of large multidomain proteins, partly because of the lack
of appropriate experimental methods to probe folding intermediates. Single-molecule force-spectroscopy
(SMFS) is particularly suited to examine folding behavior of large proteins because it can selectively
unravel arbitrary parts of the folded structure, while minimizing protein aggregation. In this report we use
AFM-based SMFS to study Firefly (Photinus pyralis) luciferase as a model for investigating the folding
behavior of large (> 500 residues), multidomain proteins. Luciferase is known to fold co-translationally
and require chaperones for refolding but its folding trajectory and the role of chaperones remain
unknown. Our results from single-molecule stretching and refolding experiments in the absence and
presence of ATP and luciferin ligands, corroborated by computer simulations show that partial unfolding
of the C-terminal residues of Luciferase - with the N-terminal domain remaining folded - allow the entire
protein to robustly refold. However, complete unfolding causes Luciferase to get trapped in mechanically
stable non-native configurations that prevent refolding, and that refolding can be rescued by adding
chaperones. These results indicate that the misfolded states only occur when C-terminal and N-terminal
unfolded residues interact. This suggests that co-translational folding is effective because it prohibits the
N-terminal residues from exploring the configurational space jointly with the C-terminal residues, and its
vectorial nature allows the N-terminal domain to fold first. This avoidance mechanism catalyzes then
folding of the middle and the C-terminal domain in Luciferase. Chaperones are not needed when only the
C-terminal residues are unfolded suggesting that they specifically assist refolding the N-terminal domain
to help the folding pathway avoid the kinetic traps.
249
POSTER ABSTRACTS
POST 07-395
Direct Measurement Of The Multimer Stabilization In The Mechanical Unfolding Pathway Of
Streptavidin
Zackary N. Scholl, Piotr Marszalek
Duke University , Durham, North Carolina, US
Understanding the stability of a protein monomer within a multimer is crucial to the development of
biopolymers and the development of protein cages for drug delivery. Here, we use single-molecule force
spectroscopy (AFM-SMFS) to measure the unfolding force of a single monomer of Streptavidin and also
measure the unfolding force of a monomer within both a tetramer and a dimer. The unfolding force
directly relates to the folding stability, and we report two main findings regarding the stability of a
monomer of Streptavidin: that is stabilized by 40% upon dimerization, and that it is stabilized an
additional 24% upon tetramerization. We also find that biotin increases stability by another 50% and that
the protein-ligand complex, rather than being strengthened through intramolecular contacts, is
strengthened due to the contacts provided by the biotin-binding loop that crosses the interface between
the dimers.
POST 07-396
ATPase domain of DnaK, Escherichia coli Hsp70 Molecular Chaperone, Experiences pH-dependent
ATPase Activity Upon Linker Binding Due to Asp194 and Glu171
Rahmi Imamoglu, Umut Gunsel, Bulent Balta, Gizem Dinler-Doganay
Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey
Hsp70 is a highly conserved molecular chaperone that play significant role in variety of cellular activities,
such as de novo folding of newly synthesized proteins, refolding of misfolded proteins, protein trafficking
and translocation to organelles. DnaK,Escherichia coli homolog of Hsp70 molecular chaperone, consists of
two domains; an N-terminal ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD),
which are connected by highly conserved hydrophobic linker. Conformational changes brought about by
substrate binding to SBD causes NBD to adopt a conformation for efficient ATP hydrolysis and in the
reverse direction ATP binding allows fast on and off rates for substrate. Previous studies showed that
389
392
allosteric communication between two domains of DnaK is provided by the conserved VLLL sequence
on the linker region. In the presence of linker, DnaK(1-392), pH-dependent higher ATPase rates are
observed, which mimics the substrate-stimulated activity of full-length protein, whereas in the absence of
linker, DnaK(1-388), behaves similar to the substrate-free unstimulated-form of the full-length. However,
it has still not been revealed which amino acids are important in the allosteric mechanism underlying the
linker binding effects to the ATPase domain. In this study, with molecular dynamic simulations, we found
out that the pH-dependent ATPase activity upon linker binding could be related to the well identified
2+
catalytic residues, Glu171, Asp194 and Asp 201, which participate in localization of Mg ion. Further pHdependent ATPase activity measurements revealed that the alkaline arm of the bell-shaped activity is
caused by a late deprotonation of Asp194 (almost 5 pH units increased pKa is observed for this Asp than
to that of the expected pKa of Asp), upon its susbstitution with Ala, alkaline site of the bell shape was
completely lost. In addition, when we mutated Glu171 to alanine we observed that the linker-bound form
of the ATPase domain did not reveal any pH-dependence. We think that linker-bound state of the ATPase
250
POSTER ABSTRACTS
domain experiences a pH-dependent ATPase activity due to a proton transfer reaction taking place during
catalytic activity and this is only occurring with linker tucking onto the ATPase domain.
Poster Session: Protein Evolution
POST 08-397
Structural Evidence for Antigen Receptor Evolution
Romain Rouet1, David Langley1, 2, Daniela Stock2, Daniel Christ1
1
Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia, 2Victor Chang Cardiac
Research Institute, Darlinghurst, New South Wales, Australia
The adaptive immune system of jawed vertebrates pre-emptively enables specific responses to pathogens
and cultivates memory of past encounters. Key components of this system are the B- and T- lymphocytes
and their “rearranging” heterodimeric receptors; the B-cell receptor (the soluble form being the canonical
“antibody”) and the T-cell receptor. Both are built from the same building block, the immunoglobulin (Ig)
domain fold. The phylogenetic pathway for the emergence of these receptors is unclear as they are
absent in the genomes of modern jawless vertebrates (hagfish and lampreys), but are present in their
jawed cousins, the sharks and rays. Hence, their emergence is believed to have occurred when these
chordate lineages diverged, some 500 million years ago. The modern heterodimeric Ig receptors are
proposed to have evolved from primordial homodimeric receptors through processes of gene duplication
and diversification. We have used laboratory evolution to reconstruct a homodimeric proto-receptor type
molecule and investigate how such a symmetric receptor is capable of interacting with protein
antigens. Crystal structures of two such Ig receptor-antigen complexes reveal that both of the Ig halves
contribute to an extensive interface capable of high affinity interactions with asymmetric
targets. Symmetry mismatch is accommodated either by the utilization of different side chain rotamers
within otherwise identical complementarity determining regions (CDRs) (conformational plasticity), or by
context-dependent differential use of CDRs (selective recruitment). These data provide structural
evidence for the evolutionary origins of modern Ig-based antibodies and receptors and support the
thesis that these molecules arose from simple Ig-domain precursors.
POST 08-398
Computational Prediction And Experimental Characterization Of A “Size Switch Type Repacking”
During The Evolution Of Dengue Envelope Protein Domain III (ED3)
Montasir Elahi1, 4, Keiichi Noguchi2, Masafumi Yohda1, Hiroyuki Toh3, Yutaka Kuroda1
1
Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and
Technology, Koganei-shi, Tokyo, Japan, 2Instrumentation Analysis Center, Tokyo University of Agriculture
& Technology, Koganei-shi, Tokyo, Japan, 3Computational Biology Research Center, AIST, Tokyo,
Japan, 4Dept. of Neurology, Graduate School of medicine, Juntendo University, Tokyo, Japan
Dengue viruses (DEN) are classified into four serotypes (DEN1-DEN4) exhibiting high sequence and
structural similarities, and infections by multiple serotypes can lead to the deadly dengue hemorrhagic
fever. Here, we aim at characterizing the thermodynamic stability of DEN envelope protein domain III
(ED3) during its evolution, and we report a structural analysis of DEN3 and DEN4 ED3 combined with a
systematic mutational analysis of residues 310 and 387. Molecular modeling based on our DEN3 and
310
387
DEN4 ED3 structures indicated that the side-chains of residues 310/387, which are Val /Ile and
310
387
Met /Leu in DEN3wt and DEN4wt, respectively, could be structurally compensated, and that a “size
251
POSTER ABSTRACTS
switch type repacking” might have occurred at these sites during the evolution of DEN into its four
serotypes. This was experimentally confirmed by a 10 ºC and 5 ºC decrease in the thermal stability of,
310
387
310
387
310
387
respectively, DEN3 ED3 variants with Met /Ile and Val /Leu , whereas the variant with Met /Leu ,
which contain a double mutation, had the same stability as the wild type DEN3. Namely, the Met310Val
mutation should have preceded the Leu387Ile mutation in order to maintain the tight internal packing of
ED3 and thus its thermodynamic stability. This view was confirmed by a phylogenetic reconstruction
310
387
indicating that a common DEN ancestor would have Met /Leu , and the intermediate node protein,
310
387
310
387
Val /Leu , which then mutated to the Val /Ile pair found in the present DEN3. The hypothesis was
further confirmed by the observation that all of the present DEN viruses exhibit only stabilizing amino acid
pairs at the 310/387 sites.
POST 08-399
Evolutionary Exploitation of Promiscuous NSAR/OSBS Enzymes
Andrew McMillan, Denis Odokonyero, Mariana Lopez, DaNae Woodard, Ashley Brizendine, Margaret E.
Glasner
Biochemistry and Biophysics, Texas A&M University, College Station, Texas, US
Many enzymes are promiscuous, catalyzing non-biological side reactions in addition to their normal
reactions. New metabolic pathways are thought to evolve by recruiting promiscuous enzyme activities. We
are evaluating this hypothesis using one of the few natural pathways that is known to use a promiscuous
enzyme. This enzyme’s ancestral activity was o-succinylbenzoate synthesis (OSBS), which is a step in
menaquinone synthesis. N-succinylamino acid racemization (NSAR) evolved in one branch of the OSBS
family, and it is used in a pathway to convert D-amino acids to L-amino acids in Geobacillus kaustophilus.
How does a single active site catalyze two reactions? Promiscuous NSAR/OSBS enzymes differ from nonpromiscuous OSBS enzymes in three ways: orientation of substrate binding, quaternary structure, and
identity of catalytically important active site residues. Second, how has the specificity of promiscuous
NSAR/OSBS enzymes evolved? Early-branching enzymes whose biological function is OSBS have minimal
NSAR activity, while late-branching enzymes whose biological function appears to be NSAR have
substantial OSBS activity. This suggests that positive selection increased the efficiency of the NSAR
reaction, but there is little disadvantage to retaining the ancestral activity. Third, how were metabolic
pathways that use NSAR activity assembled? The operon encoding the known pathway in G.
kaustophilus is poorly conserved. Instead, gene fusions and operons of some NSAR/OSBS genes suggest
that more than one metabolic pathway evolved to use NSAR activity. NSAR/OSBS genes were acquired by
diverse species through horizontal gene transfer, providing a mechanism for introducing NSAR/OSBS
genes into a variety of operons and gene fusions.
POST 08-400
Active Site Profile-Based Clustering Of Enolase Structures And Sequences
Janelle Leuthaeuser1, Stacy Knutson1, Brian Westwood1, Patricia Babbitt2, Jacquelyn Fetrow1
1
Wake Forest University, Winston Salem, North Carolina, US, 2Pharmaceutical Chemistry, University of
California - San Francisco, San Francisco, California, US
The elucidation of protein molecular function lags far behind the rate at which protein sequences are
identified; accurate and efficient computational methods that cluster protein sequences in functionally
relevant ways are essential. Active site profiling was previously developed to identify and compare details
of protein molecular functional sites. The program DASP utilizes a profile-based approach to search
sequence databases for proteins containing similar functional sites to the input proteins. The Structure
Function Linkage Database (SFLD) contains enzyme superfamilies whose functional subgroups and
families have been identified by expert curation. Therefore, validation that DASP can identify discrete
252
POSTER ABSTRACTS
functionally relevant groups corresponding to SFLD-curated groups would provide the foundation for
development of accurate and efficient computational methods to functionally cluster the protein
sequence universe. To validate the approach, active site profiles were created for each subgroup and
family in the well-studied enolase superfamily and used to search the PDB sequences. Results
demonstrate high correlation between DASP grouping and SFLD annotation, suggesting that profiling
could be used in a process to identify functionally relevant clusters, with no a priori knowledge of those
clusters. Such a process, TuLIP, was developed and applied to the enolases, producing 23 groups that
correspond well with SFLD subgroups and families. Profiles of these groups were used to search Genbank,
and over 15,500 enolase superfamily members were identified and assigned to one of the 23 functionally
relevant, discrete clusters. Continued validation and automation of this method could provide a necessary
tool to automatically cluster proteins into functionally relevant groups.
POST 08-401
Active Site Profile-Based Clustering Of The Peroxiredoxin Superfamily
Angela Harper, Janelle Leuthaeuser, Jacquelyn Fetrow
Wake Forest Univeristy, Downingtwn, Pennsylvania, US
Protein function is often predicted based on sequence similarity, leading to misannotations and
propagation of false classification of proteins. The Structure Function Linkage Database (SLFD) is
considered by many to be the gold standard for protein annotation due to manual curation. However,
this process is expensive and time-consuming, so automated processes for annotation and functional
clustering are imperative. The Peroxiredoxin (Prx) superfamily contains cysteine-based peroxidases that
react with hydrogen peroxide, organic peroxides, and peroxynitrite, and have been extensively studied by
experts making this an appropriate baseline. Using the Prx superfamily, parameters for determining
discrete and functionally relevant groups of proteins within a superfamily were defined. The Deacon
Active Site Profiler (DASP), previously developed by our lab, extracts residues around an active site
environment of a protein, creating its unique signature. Using DASP, our lab recently developed a process
called TuLIP which functionally clusters all structurally characterized members of a superfamily. The Prx
superfamily was split into four distinct TuLIP groups and the signatures from DASP for each group are
aligned and GenBank is searched for proteins with similar active site features. This is an iterative process
which uses the GenBank searches to gather information about all Prxs. The results of this process were
mapped onto a representative network of all members of the Prxs, showing that our method classified a
majority of the superfamily. This process created discrete groups in which no protein is assigned to two
different groups at significant scores. Additionally, TuLIP groups containing multiple subgroups were split
into the appropriate functionally relevant subgroups as defined by the SFLD. This method of functional
clustering is both accurate and automatable, which will aid future efforts to create a fully automated
process for annotating further superfamilies.
POST 08-402
Bioinformatics and Network Analysis of Lipocalin Superfamily
Nardos Sori, Lesley H. Greene
Old Dominion University, Norfolk, Virginia, US
Lipocalins are a superfamily of proteins that are found in various organisms with a wide range of functions
such as pheromone activity, lipid transport and coloration. Lipocalins share a common three dimensional
structure which consists of an antiparallel β-barrel and a C-terminal α-helix. Even though lipocalins are
found widely in nature, it was only recently that proteins in Wheat and Arabidopsis were identified as true
lipocalins through the elucidation of three structurally conserved regions. The study of these lipocalins is
vital as these proteins are believed to help plants tolerate oxidative stress and extreme conditions which
253
POSTER ABSTRACTS
broadens our understanding of plant sustainability in different environments. We focused on Triticum
aestivum temperature induced lipocalin (Tatil), found in wheat, an essential crop that is used by the
majority of the world population. We conducted bioinformatics studies and a network analysis of lipocalin
superfamily which sheds insight into the structure, function and evolutionary relationships of the Tatil. We
made structure-based sequence alignment of lipocalin superfamily which enabled us to calculate
conservation of the amino acid positions and elucidate a conserved long range interaction network. We
also made a model of the temperature induced lipocalin which gives us an idea of its interaction with the
cell membrane.
POST 08-403
A New Evolutionary Subclass of HMG-CoA Reductases
Jeffrey Watson, James Palmer
Chemistry and Biochemistry, Gonzaga University, Spokane, Washington, US
Isopentenyl diphosphate (IPP) is a key precursor to a diverse range of biomolecules required by all three
kingdoms of life. These molecules include prenyl groups for membrane attachment of proteins and small
molecules, plant hormones, ether-linked fatty acids in archaeal cell walls and sterols. IPP biosynthesis
proceeds via one of two known pathways. Eukaryotes, archaea and some eubacteria utilize the
mevalonate pathway, which proceeds through the action of HMG-CoA reductase (HMGR) as the ratelimiting and first committed step. Most eubacteria utilize the DXP pathway, which does not require
HMGR at any step. HMG-CoA reductase sequences were shown over a decade ago, prior to the explosion
in genome sequencing, to fall into two evolutionary classes. Class I includes eukaryotic and most archaeal
HMGRs, while Class II includes eubacterial and some archaeal HMGRs. At the time, with a single
exception, all of the eubacteria known to express HMGR also expressed the other enzymes of the
mevalonate pathway and therefore functioned biosynthetically. In contrast, HMGR from Pseudomonas
mevalonii has been shown to function in an oxidative fashion, allowing the organism to grow on
mevalonate as its sole carbon source. We have recently identified a group of bacteria that possess the
gene for HMGR, yet only have the genes for DXP pathway enzymes and not the mevalonate pathway
enzymes. One HMGR from a member of this group (Burkholderia cenocepacia) has been characterized,
and also appears to function oxidatively. Multiple sequence alignments have identified some common
characteristics of HMGRs from these organisms that suggest they represent a new evolutionary subclass
we refer to as Class II Oxidative, or Class IIox. We are employing phylogenetics, whole-genome
comparison and protein evolution tools to explore possible physiological roles for Class IIox HMGRs and
how this class of enzymes might have evolved.
POST 08-404
Ebola Protein VP35 Exploits Evolutionary Constraints In Host Defense Kinase PKR to Evade
Translational Blockade
Maayan Eitan-Wexler, Raymond Kaempfer
Department of Biochemistry and Molecular Biology, Faculty of Medicine, The Hebrew University ,
Jerusalem, Israel
Antiviral defense depends strongly on activation of the RNA-activated host kinase PKR that inhibits
translation by phosphorylating initiation factor eIF2α. To replicate, viruses must counteract PKR. The
highly lethal Ebolavirus vigorously inhibits innate immune responses through its VP35 protein. We show
that VP35 evades host defense by exploiting evolutionary constraints in PKR. PKR kinase activation
requires its homodimerization on the activating RNA. VP35 acts as a decoy of activated PKR that binds
254
POSTER ABSTRACTS
PKR, inhibiting its activation by forming a non-productive complex. VP35 mimicks the kinase activation
segment of PKR wherein two highly conserved threonines must undergo phosphorylation to enable PKR
activation and eIF2α substrate recognition, both replaced in VP35 by phosphomimetic residues.
Mutation of these phosphomimetic residues sufficed to abrogate the PKR-antagonist activity of VP35. By
mimicking activated PKR, the kinase-dead VP35 protein presents a unique viral strategy to evade hostmediated translational blockade. We thus demonstrate a novel molecular mechanism of immune evasion
and show that evolutionary constraints can render a host target protein powerless vis-à-vis a pathogen.
POST 08-405
The Substrate Specificity “Lock;” Evolutionary Epistasis In Apicomplexan Lactate And Malate
Dehydrogenases
Brian Beckett1, Michelle Y. Fry2, Douglas L. Theobald1
1
Biochemistry and Biophysics, Brandeis University, Waltham, Massachusetts, US, 2Biophysics, Brandeis
University, Waltham, Massachusetts, US
The unusual Apicomplexan lactate dehydrogenase (LDH) evolved from a malate dehydrogenase (MDH)
via a six amino acid insertion in the active site loop, which shifts specificity from malate to lactate by over
twelve orders of magnitude. When these six amino acids are inserted in the active site loop of the modern
MDH, all enzyme activity is lost. However, in ancestrally reconstructed MDH the same insertion creates a
highly-active bifunctional enzyme. Evolution since the ancestor has resulted in mutations that “lock in”
substrate specificity; residues distal to the active site must have non-trivial epistatic interactions that are
changing throughout evolution. To determine the nature of the “lock” in substrate specificity, we
generated ancestral intermediates along the Cryptosporidium parvum lineage. We cloned, expressed, and
purified the ancestors in E. coli., and characterized their substrate specificities using steady-state
Michaelis-Menten kinetics. The number of potential residues responsible for epistasis was narrowed using
site-directed mutagenesis. Our data suggest that substrate specificity was only recently “locked” in the C.
parvum family tree. The epistatic changes during evolution may be dynamic inter-residue interactions,
which can not be observed using x-ray crystallography. Therefore, we generated mutant MDH and LDH
amenable to nuclear magnetic resonance (NMR) characterization. Preliminary NMR spectra have been
obtained for ancestral MDH. Future NMR studies will inform the dynamics of how epistasis has evolved to
lock substrate specificity in Apicomplexa.
POST 08-406
Multi-level Iterative Functional Clustering of Glutathione Transferase Superfamily
Kiran Kumar, Janelle Leuthaeuser, Brian Westwood, Jacquelyn Fetrow
Physics, Wake Forest University, Winston-Salem, Virginia, US
High throughput sequencing has resulted in a significant misannotation increase within large protein
databases such as GenBank. These incorrect classifications are often due to function annotation at a level
beyond what the data currently suggests. Relying on manual curation, however, to categorize the high
volume of sequence data is neither cost nor time effective. Therefore, it is imperative to develop
automated methods for functionally classifying proteins. To meet this need, our lab is developing
parameters for a computational, multi-level, iterative searching procedure to functionally cluster protein
sequences. Instead of full sequential or structural analysis, this procedure clusters proteins based on the
structural and sequential information within the active site. To test the parameters of this procedure, The
Structure Function Linkage Database (SFLD) is used as a gold standard because it contains expertly
curated proteins. The Glutathione S-Transferase superfamily, in particular, is being used to explore our
ability to match SFLD GST subgroup designations. First, the GST structures are clustered into distinct
255
POSTER ABSTRACTS
groups based on active site similarity. The Deacon Active Site Profiler (DASP), previously developed in our
lab, searches GenBank using these groups to generate hits of functionally analogous proteins. Hits can be
tracked onto a map of all the GST sequences to visually assess the progression of expanding an initial
search, which used only structurally characterized GST members, to the mostly uncategorized sequential
universe of the entire superfamily. Results have shown that our methods are successful in matching SFLD
annotation in the majority of clusters when searching GenBank. Findings also indicate the potential to
extend family level annotation to GST proteins, which have not yet been determined by the SFLD. Overall,
results demonstrate that current parameters produce functionally relevant GST clusters, which indicates
the feasibility of developing an automated, accurate method to functionally cluster protein superfamilies.
POST 08-407
Structural Analysis of Toxin/immunity Complexes in Contact-Dependent Growth Inhibition Systems
Parker M. Johnson1, Robert P. Morse1, David A. Low2, Christopher S. Hayes2, Celia W. Goulding1
1
Molecular Biology and Biochemistry, UC Irvine, Irvine, California, US, 2Molecular, Cellular, and
Developmental Biology, UC Santa Barbara, Santa Barbara, California, US
Bacteria have evolved complex strategies to compete and communicate with one another. A new
mechanism of interbacterial competition, termed contact-dependent growth inhibition (CDI) was recently
1,2
discovered . CDI systems are found in a wide variety of gram-negative bacteria, including many
1
important human pathogens such as uropathogenic E. coli strain 536 (UPEC536) and Burkholderia
pseudomallei3. CDI allows for competitive advantage in the ability to kill neighboring bacterial cells
mediated by contact of the CdiA protein with a target cell, where its C-terminal domain (CdiA-CT) is
translocated into the target cytosol and inhibits growth of non-cognate target cells. In order to prevent
+
susceptibility to their own toxins, CDI bacteria express a cognate immunity protein (CdiI) to specifically
2
bind and inactivate CdiA-CT . Burkholderia pseudomallei environmental isolates E479 and 1026b have
3
been shown to utilize CDI mechanisms to outcompete neighboring bacteria , signifying the pathway’s
importance in growth and survival. The toxin and immunity proteins between these two isolates share
significant sequence identity, and both toxins exhibit tRNase activity; however the toxins have different
tRNA specificities along with cleavage sites, and cognate immunity proteins only bind to cognate
toxins. We have solved the structures of these two B. pseudomallei toxin/immunity complexes that shed
light on toxin specificity as well as the unique toxin/immunity protein interfaces. The CDI system from
UPEC536 is unusual, CdiA-CT exhibits toxic tRNase activity only when activated by binding target cell
4
CysK, a permissive factor involved in cysteine biosynthesis . We have solved the X-ray crystal structure of
the toxin/immunity/CysK complex to 2.99 Å. We will discuss the interaction of CysK with toxin and its
neutralization by the immunity protein, and the functional implications gleaned from this ternary
complex. 1. S.K. Aoki et al., Science 309, 1245 (2005). 2. S.K. Aoki et al., Nature 468, 439 (2010). 3. K.
Nikolokakis et al., Mol Microbiol. 84, 516 (2012). 4. E.J. Diner et al., Genes & Dev 26, 515 (2012).
POST 08-408
Automated Functional Clustering of the Crotonase Superfamily based on active site motif
sequences
Julia Hayden, Janelle Leuthaeuser, Brian Westwood, Jacquelyn Fetrow
Wake Forest University , New Cumberland, Pennsylvania, US
The increasing ease of protein sequencing has lead to heightened use of computational methods
utilizing full protein sequences to rapidly classify the growing number of proteins, leading to rampant
misannotation in databases using these computational methods. Manually curated databases, such as
256
POSTER ABSTRACTS
the The Structure Function Linkage Database (SFLD) have less annotation errors and can be used as a
gold standard. One of the superfamilies in the SFLD, the Crotonase superfamily, contain proteins related
by conserved peptidic NH groups that mediate the formation of an oxyanion hole. A process formerly
completed, termed TuLIP, iteratively clustered 100 non-redundant structurally represented proteins
based on active site from the Crotonase superfamily into 22 distinct protein groups through use of
DASP, a program written to compare and score similarity between protein active sites. For all
structurally characterized proteins that did not cluster with any groups, a method of profile engineering
was utilized to take advantage of structural information to find other sequences with similar active sites.
The 22 groups were used in isolated searches of NR Genbank via protein active sites to find other
functionally similar proteins. Protein hits resulting from these searches were examined by similarity
score and on family level based on the SFLD hierarchy, and plotted on a representative network of
proteins in the Crotonase superfamily to examine coverage. This search process showed high correlation
with the data presented in the SFLD with protein groups highly discreet at a DASP score cut-off of 1E-12
as well as high coverage of the Crotonase superfamily representative network. The results of this
process indicate a step forward in creation of an accurate computational method to separate proteins
into functionally related groupings.
POST 08-409
Domain Exchange Between Membrane And Soluble Proteins Contributes To Expand Intercellular
Communication Network
Hyun Jun Nam1, Inhae Kim2, James U. Bowie3, Sanguk Kim1, 2
1
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Korea, Republic
of, 2Department of Life Sciences, POSTECH, Pohang, Korea, Republic of, 3Department of Chemistry and
Biochemistry, UCLA, Los Angeles, California, US
The transition to multicellularity from protozoa marks one of the most pivotal and poorly understood
events in life’s history. We hypothesize that membrane proteins must be key players in the development
of multicellularity because they are well positioned to form the cell-cell contacts and to provide the
intercellular communication required for the creation of complex organisms. We found that a major
mechanism for the necessary increase in membrane protein complexity during the transition to
multicellularity was the new incorporation of domains from soluble proteins. Moreover, the membrane
proteins that have incorporated soluble domains in metazoans are enriched in the functions unique to
multicellular organisms such as cell-cell adhesion, immune defense and developmental processes. They
also show enhanced protein-protein interaction (PPI) network complexity and centrality, suggesting an
important role in the cellular diversification found in complex organisms. Our finding provides empirical
evidence for metazoan evolution process that highlights the importance of domain recombination
between membrane and soluble proteins to expand intercellular communication network.
POST 08-410
Utilization of an Iterative Clustering Method to group Radical SAMs in Functionally-Relevant Ways
Gabrielle B. Shea, Janelle B. Leuthauser, Brian Westwood, Jacquelyn S. Fetrow
Wake Forest University, Remsenburg, New York, US
Over the past decade, the use of computational techniques has greatly increased the amount of data
gathered on protein sequence, structure, and function; however, this data is often poorly analyzed,
resulting in a greater prevalence of functional misannotations. These misannotations make it difficult to
identify relationships between proteins, and understanding these relationships is highly useful when
attempting to identify new candidates for drug therapies. Although manually-curated databases like the
Structure-Function Linkage Database (SFLD) are nearly free of misannotations, hand curation is neither
257
POSTER ABSTRACTS
cost- nor time-effective. Our lab is developing a Function Annotation Protocol (FAP) that utilizes
computational tools to accurately and efficiently classify proteins based on active site information. This
iterative pipeline uses structural information to cluster the proteins in an SFLD-defined superfamily into
smaller, functionally-relevant groups. This project focuses on the Radical SAMs, a superfamily of
proteins for which there are many more known subgroups and families than there are structures. The
goal of the project is to use the active site environment to compare proteins and to search databases in
order to find and annotate all of the sequences in this superfamily. We have already used the Deacon
Active Site Profiler (DASP), a computational tool developed by our lab, to cluster Radical SAM protein
structures into groups that can be deemed functionally relevant based on active site similarity. For
those structural representatives that didn’t fit in any group, we engineered a group based on proteins
with high sequence similarities. However, DASP has not yet hit all of the proteins that the SFLD claims
are members of the Radical SAM superfamily because most of the members do not have known
structures, and DASP relies on structural inputs. Thus, this project aims to subcluster in the initial results
of GenBank searches in order to create more specific groups of proteins that can therefore be used by
DASP to pick up the Radical SAM protein sequences that are more distantly related to the original
groups of protein structures.
POST 08-411
Directed Evolution of Duplicated Qbeta RNA replicases in liposome
Keisuke Uno1, Takeshi Sunami1, 3, Yasuaki Kazuta3, Norikazu Ichihashi1, 3, Tetsuya Yomo1, 3, 2
1
Graduate School of Information Science and Technology, Osaka University, Osaka, Japan, 2Graduate
School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan, 3YOMO Dynamical Micro-scale
Reaction Environment, ERATO, JST, Suita, Osaka, Japan
Gene duplications play an important role in the evolution of novel gene functions. Many research of gene
duplications are based on genomic sequence analysis and population modeling. There is a few research of
experimental evolution and analysis of its evolutionary process. We have tried experimental directed
evolution of duplicated genes in vitro, and analyzed the functional changes of the two
genes. “Translation-coupled RNA replication (TcRR) system” (H. Kita, 2008), in which the genetic
information (RNA) is replicated by self-encoded replicase, was used as a model reaction for the directed
evolution. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA
that encodes the protein, and the replicase replicates the template RNA. To detect the RNA replication,
we used a reporter RNA (GUS(-) RNA) which encodes an antisense sequence of beta-glucuronidase (GUS)
gene. After replicase synthesis in TcRR system, the complementary strand of the reporter RNA (GUS(+)
RNA) is synthesized by the replicase, and fluorogenic substrate is hydrolyzed by GUS translated from the
GUS(+) RNA . “Liposome-based IVC” (T. Nishikawa, 2012) was used as a method for the directed
evolution. Briefly, a DNA library of duplicated and mutated beta-subunit of replicase was
compartmentalized into liposomes. After 3 hours reaction, liposomes exhibiting the strongest
fluorescence signals were sorted by a fluorescence-activated cell sorter. The genes obtained were
amplified in a test tube, and transferred to the second round of screening. The DNA library after 5th
selection round (G1R5) showed about 3 times higher reaction activity than original DNA (WILD). For next
round, G1R5 was mutated by error-prone PCR to G2R1. G2R9 showed about 6 times higher reaction
activity than WILD after 9th selection round. We have showed that the duplicated genes can evolve using
liposomes as microreactors.
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POSTER ABSTRACTS
POST 08-412
Comparative Analysis of Post-translational Modification Hot Spots in Phylogenetically Distant
Heterotrimeric G proteins
Shilpa Choudhury, Henry Dewhurst, Matthew P. Torres
G protein signaling (GPS) systems composed of G protein coupled receptor (GPCR) and heterotrimeric G
proteins (Gαβγ) are accountable for transducing a myriad of extracellular stimuli including light,
neurotransmitters and hormones. Emerging evidence suggests that heterotrimeric G proteins are
regulated by a variety of post-translational modifications (PTMs). Recent bioinformatics analyses of
mammalian G protein PTMs using an in-house toolkit (Structural Projection of PTMs (SPoP)) shows that
Gα, Gβ and Gγ each exhibit PTM “hot spots” within their respective structures. These hot spots are most
frequently encountered at GPS protein interfaces or in catalytic regions, suggesting that they play an
important role in regulating signaling. Here, we tested the hypothesis that similar hot spots would be
modified in the phylogentically distant Saccharomyces cerevisiae, which harbors a single canonical GPS
system sensitive to mating pheromones. To test the hypothesis, we identified PTMs on each of the yeast
G proteins using affinity purification mass spectrometry (AP-MS). Since yeast G proteins are highly
modified under conditions of GPCR activation or nutrient stress, we assayed each G protein under both
conditions independently. AP-MS resulted in identification of all modification sites currently known as
well as additional sites of modification. Phosphorylation is the predominant PTM found on yeast G
proteins, with the Gα subunit (Gpa1) exhibiting the greatest number of identified sites. While Gpa1 was
highly phosphorylated at multiple sites under nutrient stress conditions (low glucose), multi-site
phosphorylation of Gβ (Ste4) was stimulated upon GPCR activation with mating pheromone. To estimate
conservation of structural PTM hotspots between yeast and mammals, we performed predictive
structure threading of yeast G proteins, and compiled the AP-MS results into a structural topology. We
then surveyed the degree of overlap between yeast and mammalian G protein topologies using a range
of criteria including conservation of modified amino acid residue, structure, and protein interface
residence. We find that residues involved in guanine nucleotide binding and in interactions between G
protein subunits are structurally conserved PTM hotspots. These data suggest the existence of a
conserved mechanism of regulation for G protein signal transduction by post-translational modification
of heterotrimeric G proteins.
POST 08-413
The Evolution Of Caffeine Synthases In Theobroma Cacao
Craig D. Thulin
Chemistry, Utah Valley University, Orem, Utah, US
How novel protein-coding genes evolve is a question of great interest. The accepted paradigm is that of
gene duplication followed by divergent mutations that lead to the sequence of a new protein with new
function. An example of this mechanism of protein evolution was sought among the caffeine
synthases. Several caffeine synthases have been characterized from Coffea (coffee) and Camellia (tea)
plants. These appear to be the results of gene duplication events because all of the caffeine synthases are
closely related within (but not between) each of these two somewhat distantly related genera. One gene
encoding caffeine synthase has been characterized from the cocoa plant Theobroma cacao, which does
not closely cluster with either Coffea or Camellia caffeine synthases. The genome of T cacao has been
sequenced, and a number of closely related putative caffeine synthase genes are found in the genomic
area on chromosome ten where the known caffeine synthase gene is located. We find that elsewhere in
the T cacao genome there are four other genes encoding more distantly related N-methyltransferase
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POSTER ABSTRACTS
genes. Comparison of non-coding regions of these genes with the genes on chromosome ten enables us
to deduce a probable evolutionary history of the caffeine synthase genes in T cacao, which do appear to
be the result of gene duplication followed by divergence.
POST 08-415
Quantitative Imaging with Amersham™ Imager 600
Erik Bjerneld, Erika Svensson, Johan Johansson, Maria Winkvist, Hakan Roos
GE, San Francisco, California, US
Electrophoretic based protein- and DNA separation are well established techniques that are widely used
in Life Sciences. However, the development of highly sensitive detection reagents together with
advanced imaging techniques has shifted the obtained results from qualitative towards quantitative. To
obtain quantitative results, the whole workflow has to be considered. Here we have focused on the
importance of the CCD imager system, suitable applications and important considerations when working
quantitatively. The Amersham Imager 600 QC or RGB, which is autocalibrated for accurate and reliable
measurements of optical density, generates linear quantitative measurements of proteins stained with
colorimetric stains such as Coomassie or silver. Amersham Imager 600 QC is a dedicated configuration
for densitometry applications in a QC environment. Quantitative Western blotting requires a signal
response that is proportional to the amount of protein. A broad dynamic range with linear response
allows you to simultaneously quantitate both high and low levels of proteins with a signal response that
is proportional to the amount of protein. Furthermore, the minimal crosstalk of Amersham Imager 600,
and the spectrally resolved dyes Cy™2, Cy3, and Cy5, makes it a suitable system for a wide range of
multiplexing applications, such as the detection of several proteins at the same time or different
proteins of similar size.
POST 08-416
Solution NMR Structure of the SLED Domain from Scml2 (Sex Comb on Midleg-like 2) - a New
Domain on Epigenetic Landscape.
Irina Bezsonova
University of Connecticut Health Center, Farmington, Connecticut, US
Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human
Scml2 is a part of a multi-subunit protein complex, PRC1, which is responsible for maintenance of gene
repression, prevention of chromatin remodeling, preservation of the “stemness” of the cell and cell
differentiation. While the majority of PRC1 subunits have been recently characterized, the structure of
Scml2 and its role in PRC1-mediated gene silencing remain unknown. We have identified a conserved
protein domain within human Scml2 and determined its structure by solution NMR spectroscopy. We
named this module Scm-Like Embedded Domain, or SLED. Evolutionarily, the SLED domain emerges in the
first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is
exclusively found within the Scm-like family of proteins, often accompanied by MBT and SAM domains.
The domain adopts a novel alpha/beta fold with no structural analogues found in the Protein Data Bank.
We also examined the ability of the SLED domain to bind double-stranded DNA, and show that the
isolated domain interacts with DNA in a sequence-specific manner. Since PRC1 complexes localize to the
promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an
important link connecting the PRC1 complexes to their target genes
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POSTER ABSTRACTS
POST 08-417
Monitoring the Effect Of Extensive Genetic Drift on the Evolution Of Novel Enzyme Function
Linda Jäger, Peter Kast, Donald Hilvert
ETH Zürich, Zürich, Switzerland
Enzymes evolve through neutral genetic drift creating a panoply of different proteins with varying
stabilities and promiscuous activities. It is conceivable that today’s spectrum of highly active catalysts
arose from the natural selection of such promiscuous precursor proteins. Here we chose to study EcCM,
the chorismate mutase (CM) domain of the Escherichia coli P-protein, responsible for the pericyclic
rearrangement of chorismate into prephenate, a branchpoint reaction in the biological synthesis of
phenylalanine and tyrosine. Interestingly, EcCM strongly resembles PchB, an enzyme from Pseudomonas
aeruginosa, in its structure, active site organization, and transition state analog binding. PchB is an
isochorismate pyruvate lyase (IPL), catalyzing the concerted conversion of isochorismate to salicylate
and prephenate. While PchB has some residual CM activity in addition to its main function, EcCM shows
no promiscuous IPL activity. These observations jointly suggest that both enzymes stem from a
common ancestor. Nevertheless, decades of research employing state-of-the art protein engineering
strategies have failed in creating IPL activity in EcCM. Therefore, we chose to recapitulate in the lab by a
neutral drift experiment what presumably has occurred in nature before. We drifted EcCM and PchB
over 20 generations of error-prone PCR mutagenesis followed by low-stringency selection for CM
activity to maintain the overall structural fold thereby increasing the likelihood of finding novel catalysts.
The 107-108 variants generated this way each contained 1-25 nucleotide mutations (up to 10% of the full
length gene) thus representing a significantly genetically diversified population considering the low
spontaneous mutation rates acting on contemporary genomes (ranging in the order of 10-9 substitutions
per base per bacterial replication cycle). Initial data suggest that the genetic drift of either enzyme
predominately decreases the native catalytic efficiency while possibly promoting increased substrate
promiscuity. Deep sequencing experiments are currently underway and are expected to provide further
clues as to which residues to target for further biochemical characterization of their prospective CM or
IPL activity.
POST 08-418
Synuclein and the Coelacanth
James M. Gruschus
NHLBI, NIH, Bethesda, Maryland, US
The alpha-synuclein member of the synuclein family appears to have arisen in an early Sarcopterygian, the
common ancestor of land vertebrates and the coelacanth, a lobe-finned fish. Compared to ray-finned
fish, the sequences of alpha- and beta-synuclein show significantly more conservation among land
vertebrates and coelacanths, suggesting they have evolved more important biological roles involving
additional functions and/or interaction partners. Two potential partners of alpha-synuclein are
synaptobrevin-2 (VAMP-2), part of the SNARE complex involved in presynaptic vesicle release, and
glucocerebrosidase (GCase), a lysosomal enzyme in which mutations have been linked to Parkinson
disease. If these proteins are truly biological partners of alpha-synuclein, then they must have co-evolved,
that is, their interaction must be subject to evolutionary selection. To test the co-evolution hypothesis,
correlated mutation analyses of alpha-synuclein with VAMP-2 and GCase have been performed using up
to 57 species, including mammals, birds, reptiles, amphibians and coelacanth. While the number of
species is too few to generate robust correlations via the analyses alone, when combined with
experimental results a number of intriguing correlated mutations emerge. For instance, the highest
correlations with VAMP-2 occur in its first 28 residues, truncation of which is known to result in loss of
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POSTER ABSTRACTS
interaction with alpha-synuclein. For GCase, the correlation between H374 and alpha-synuclein E132
could explain why alpha-synuclein only interacts with GCase at the acidic pH found in lysosomes. This
study highlights the benefits of combining bioinformatics with experiment, a partnership whose
importance will continue to grow as more and more genomes are sequenced.
Poster Session: Proteins in Altered States
POST 09-419
Storage of Environmental Information into Biological Structures: Temperature-dependent Fibrillar
Polymorphism of α-Synuclein
Ghibom Bhak, Seung Ryeoul Paik
Seoul National University , Seoul, Korea, Republic of
Molecular-level storage of environmental information into tangible structures and their subsequent
inheritance to next generations have been studied with a biological phenomenon of amyloidogenesis.
Amyloidogenesis defines a biochemical condition producing highly ordered amyloid fibrils from soluble
proteins via molecular disorder-to-order transition. α-Synuclein is an amyloidogenic protein responsible
for the Lewy body formation in Parkinson’s disease (PD). Based on the accelerated amyloidogeneses of αsynuclein in its oligomeric state with shear force and hexane, we have proposed a double-concerted
fibrillation mechanism where two successive concerted assemblies of monomers and oligomers have led
to the amyloid fibril formation. By employing the α-synuclein oligomers as an environment sensing agent,
an intangible information of temperature has been stored into the tangible supra-structures of amyloid
fibrils in various morphologies through the oligomeric unit-assembly process. The stored information,
then, has been perpetuated by self-propagating the diversified fibrillar structures with distinctive
molecular characteristics to next generations through the nucleation-dependent fibrillation process. This
oligomer-mediated molecular inscription process of environmental information, therefore, may not only
represent influx of information into biological system but also be applied in the areas of
nanobiotechnology to process various external signals.
POST 09-420
Reversible Polymeric Fibers made from Low-Complexity Sequences Function as a Foundation of
RNA Metabolism
Masato Kato, Steven McKnight
Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, US
Low-complexity (LC) sequences have been believed to perform important biological roles by unknown
mechanisms. We have discovered that purified liquid samples of the LC domains of RNA-binding
proteins, such as FUS, hnRNPA2 and TDP-43, made the transition into a gel-like phase in a
concentration-dependent manner1,2. The hydrogels are composed of morphologically uniform polymers
endowed with an underlying cross-β structure. Unlike pathogenic amyloid fibers, our polymeric fibers
are reversible. Hydrogel has capability to capture LC domains of RNA granule proteins via copolymerization of the LC domain forming the hydrogel and the test LC domain into a single polymeric
fiber. The C-terminal domain (CTD) of RNA polymerase II3 and SR domains of splicing factors also bind to
the hydrogel, yet these interactions are reversible by phosphorylation of CTD and SR domains by their
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POSTER ABSTRACTS
respective kinases. The outcome of these studies has led to a conceptual framework hypothesizing the
structural underpinnings of how dynamic puncta, including RNA granules, transcription factories and
nuclear speckles, form in eukaryotic cells. Many mutations in these LC domains found in ALS and FTLD
patients have been reported to facilitate fibril aggregate formation of these proteins and thereby to
possibly exert toxicity on neuronal cells. We hypothesize, as mechanisms for the aggregate formation,
that disease-causing mutations in LC domains tip the equilibrium of reversibility of polymeric fiber
formation such that the fibers become unacceptably stable, resulting in accumulation of irreversible
polymers. References 1. Kato, M., Han, T. W., Xie, S., Shi, K., Du, X., Wu, L., Mirzaei, H., Goldsmith, E.,
Longgood, J., Pei, J., Grishin, N., Frantz, D., Schneider, J., Chen, S., Li, L., Sawaya, M., Eisenberg, D., Tycko,
R. and McKnight, S. L. Cell-Free Formation of RNA Granules: Low Complexity Sequence Domains Form
Dynamic Fibers Within Hydrogels, Cell 149,753-767 (2012) 2. Han, T. W., Kato, M., Xie, S., Wu, L.,
Mirzaei, H., Pei, J., Chen, M., Xie, Y., Allen, J., Xiao, G. and McKnight, S. L. Cell-Free Formation of RNA
Granules: Bound RNAs Identify Features and Components of Cellular Assemblies, Cell 149, 768-779
(2012) 3. Kwon, I., Kato, M., Xiang, S., Wu, L., Theodoropoulos, P., Mirzaei, H., Han, T., Corden, J. L. and
McKnight. S. L. Phosphorylation-regulated Binding of RNA Polymerase II to Fibrous Polymers of LowComplexity Domains, Cell 155, 1049 (2013)
POST 09-421
Flavone Derivatives As Inhibitors Of Insulin Amyloid-Like Fibril Formation
Ricardas Malisauskas, Akvile Botyriute, Vytautas Smirnovas
Vilnius University Institute of Biotechnology, Vilnius, Lithuania
A number of diseases are associated with formation of amyloid fibrils. Both, mature fibrils and oligomers
or protofibrils, which can exist on pathway of fibril formation, may be responsible for the pathogenesis.
Thus any molecules, able to change kinetics or alter the pathway of protein aggregation, can potentially
interfere with diseases. A number of compounds, including several natural and synthetic flavone
derivatives were reported as inhibitors of amyloid fibril formation. Moreover, it was reported that the
inhibition potential depends on the number and the position of hydroxyl groups across the flavone
backbone. In most of studies the main value used to rate the inhibition potential of flavones is
fluorescence intensity of Thioflavin T (ThT) binding assay. We studied impact of 260 commercially
available flavone derivatives on insulin fibril formation. In addition to ThT fluorescence intensity we also
compared halftime of aggregation (t50). Almost one third of studied flavone derivatives changed final ThT
fluorescence at least two fold, however most of these derivatives had much smaller impact on t50, with no
clear correlation between these two values.
POST 09-422
Phosphorylation Releases Constraints to Domain Motion in ERK2
Yao Xiao1, Thomas Lee1, 2, Michael P. Latham1, 4, Lisa R. Warner1, 5, Akiko Tanimoto1, 6, Arthur Pardi1,
Natalie G. Ahn1, 2, 3
1
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, US, 2HHMI,
University of Colorado, Boulder, Colorado, US, 3BioFrontiers Institute, University of Colorado, Boulder,
Colorado, US, 4Department of Molecular Genetics, University of Toronto, Toronto, Ontario,
Canada, 5Institute of Structural Biology, Neuherberg, Germany, 6Department of Chemistry and
Biochemistry, Ohio State University, Columbus, Ohio, US
Protein dynamics has been shown to be important for regulating protein function, such as allostery and
enzyme catalysis. It remains a challenging question what is the direct role of protein dynamics in enzyme
catalysis. Here, we present NMR data on millisecond ILV methyl dynamics of MAP kinase ERK2 in its
inactive and active forms, using 13C relaxation dispersion experiments. ERK2 is activated by dual263
POSTER ABSTRACTS
phosphorylation. Inactive, unphosphorylated ERK2 shows only local ILV methyl dynamics. Upon
activation, the dispersion data demonstrate that ERK2 undergoes global conformational dynamics. The
observed dynamics in active ERK2 is consistent with inter-conversion between two major conformations
that affect residues throughout the kinase core, with kex ≈ 300 s-1 (kAB≈240 s-1/kBA≈60 s-1) and pA/pB ≈
20%/80%. A mutant of ERK2 was designed to increase backbone flexibility at the hinge region, the pivot
point connecting N- and C-terminals. The methyl dynamics data of this mutant of inactive ERK2, also
indicate inter-conversion between conformations, with kex ≈ 500 s-1 (kAB≈15 s-1/kBA≈485 s-1) and pA/pB ≈
97%/3%. Thus, activation of ERK2 by phosphorylation leads to a dramatic shift in global conformational
exchange, which is likely through release of constraints at the hinge.
POST 09-423
Using Mass Spectrometry To Define How The Small HSP Chaperones Protect Substrates From
Aggregation
Keith Ballard2, Heather O'Neill1, Wenzhou Li1, Vicki Wysocki3, Elizabeth Vierling2
1
Department of Chemistry and Biochemistry, University of Arizona, Tuscon, Arizona, US, 2Biochemistry
and Molecular Biology, University of Massachusetts, Amherst, Amherst, Massachusetts, US, 3Chemistry
and Biochemistry, Ohio State University, Columbus, Ohio, US
Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent
molecular chaperones linked to diseases of protein misfolding. They comprise a conserved core αcrystallin domain (ACD) flanked by an evolutionarily variable N-terminal arm (NTA) and semi-conserved Cterminal extension. sHSPs are capable of binding up to an equal mass of unfolding protein, forming large,
heterogeneous sHSP-substrate complexes that make substrate available to the ATP-dependent
chaperones for refolding. Understanding sHSP-substrate interactions may provide insight into developing
potential therapeutic strategies for treating protein-folding diseases such as Alzheimer’s and Parkinson’s
disease. To derive common features of sHSP-substrate recognition, we compared the chaperone activity
and specific sites of interaction with substrate for three different sHSPs: an unusual dimeric sHSP from
Arabidopsis, At18.5, and more typical oligomeric sHSPs from pea (Ps18.1) and wheat (Ta16.9), for which
the atomic structure is available. We used the homobifunctional amine-reactive, 7 Å crosslinker
2
bis(sulfosuccinimidyl)glutarate (BS G) to test for interaction sites between each of the three sHSPs and the
model substrate malate dehydrogenase (MDH), identifying crosslinking by mass spectrometry using an
LTQ-Orbitrap. MDH exhibits major rearrangement upon heat-denaturation and association with sHSPs as
evidenced by detection of new MDH-MDH crosslinks incompatible with the MDH native structure. The
NTA of all three sHSPs was a major site of crosslinking to substrate, and was found linked to multiple Lys
residues on MDH, consistent with the heterogeneity of the sHSP-MDH complexes. Not all sHSP Lys
residues were involved in crosslinking, although they reacted with the crosslinker, demonstrating
preferred sites of interaction. In total, the data support an important role for the NTA in substrate
recognition and indicate that an early unfolding intermediate of the substrate is recognized.
POST 09-424
Mechanically resistant conformations in amyloid β and α-synuclein
Sigurdur Æ. Jònsson2, Simon Mitternacht1, Anders Irbäck2
1
Science Library, University of Bergen, Bergen, Norway, 2Astronomy and Theoretical Physics, Lund
University, Lund, Sweden
Unstructured proteins such as the amyloid β-peptide (Aβ) and α-synuclein (αS) display remarkable
mechanical resistance in a significant fraction of unfolding traces in single-molecule pulling experiments.
The rupture forces are comparable to those of mechanically stable folded proteins. The nature of these
force-resistant states, and their relation to amyloid aggregation is unknown. We have performed all264
POSTER ABSTRACTS
atom pulling simulations on broad ensembles of Aβ and αS conformations to identify the mechanically
resistant states. The simulated unfolding trajectories have rupture events similar to experiments both in
molecular extension and distribution of rupture forces. We find that the most resistant states share a
common architecture, which in both cases can be related to that of amyloid fibrils. We also find that the
disease-linked arctic mutation of Aβ increases the occurrence of highly force-resistant structures. We
propose that similar structures cause the high experimental rupture forces and that they could play an
important role in the amyloid aggregation pathway.
POST 09-425
Structural Determinants of Amyloid Fibril Formation in Triosephosphate Isomerase.
Edson N. Carcamo-Noriega, Gloria Saab-Rincón
IBT-UNAM, cuernavaca, Mexico
The amyloid fibril is one of the most biologically important protein structures due to its implication in
numerous degenerative diseases. Although all proteins have the potential to form these aggregates,
because their formation relies on main chain interactions, not all of them can form fibrils under
physiological conditions. The structural determinants that promote or inhibit the formation of amyloid
fibril remain unknown. This study aims to find these determinants in the human enzyme triosephosphate
isomerase, whose ability to form fibrils according to recent reports may be associated with Alzheimer's
disease. Aggregation kinetics under destabilizing conditions followed by thioflavin T show that
triosephosphate isomerase form cross-beta structures reaching saturation within 72 hrs of incubation. The
presence of a cross-beta core in non-fibrillar morphology was also confirmed with the antibody WO1 in
dot-blot assays and fluorescence microscopy. The cross-beta region was found by a prediction analysis
using several predictors. The three regions with the highest score were synthesized and tested under
physiological conditions. The region 183-213 was able to form fibrils confirmed with thioflavin T binding,
WO1 dot-blot assay and transmission electron microscopy. Despite the presence of a cross-beta region
within the structure of triosephosphate isomerase the enzyme formed non-fibrillar aggregates, this may
be due to protection by stable modules βα adjacent to the region cross-beta that could work like steric
hindrance, preventing the elongation of the fibril and stopping the aggregation in a protofibrillar state.
POST 09-426
Developing Quantitative NMR Methods For Predicting Residue Specific Helicity Of MetO-λ
Unfolded State
Kan Li, Roy Hughes, Terrence G. Oas
Biochemistry, Duke University, Durham, North Carolina, US
Numerous studies have provided evidences for residual compactness in protein unfolded state. Studies
of unfolded state conformational ensemble will greatly contribute to understanding the starting point of
protein folding reaction. NMR techniques are powerful in detecting residue specific information.
Nascent helicity, local hydrophobic collapse and long range interactions have been observed in the
unfolded state of several proteins by NMR techniques.
We use λ repressor N-terminal domain, a
helical protein, to study mainly the residual helicity of the unfolded state ensemble along with other
possible structural preferences. A variant of the λ repressor N-terminal domain has been constructed by
oxidizing protein core methionine residues along with other mutations (MetO-λ), allowing it to populate
unfolded state ensemble under nondenaturing conditions. NMR backbone dynamics study of MetO-λ
unfolded state shows residual helicity and possible hydrophobic collapse. In order to develop methods
to quantitatively determine residue specific helicity of MetO-λ unfolded state, we expressed residue 830 of λ repressor protein as an ioslated peptide (LRH1x), which corresponds to extended first helix of the
protein. The first helix is important in domain folding and intrinsically helical in isolation. Analysis of CD
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POSTER ABSTRACTS
spectra and backbone chemical shift (CS) indices of LRH1x at neutral pH both show evidences of
significant helicity. However, to predict residue specific helicity, we developed a complementary method
in addition to CS indices method due to its intrinsic limitation on the quality of prediction.
The
protection factors (PFs) of backbone amide hydrogens contain averaged information of residue specific
helicity. By comparing PFs from measured amide proton exchange rates and theoretically calculation
based of helix-coil transition theory, we are aiming to use quantitative residue specific observation to
optimize the helix-coil transition theory to better prediction and understanding of residue specific
helicity. We implemented this method for LRH1x and was able to observe discrepancies between
experimentally and theoretically extracted PFs, which indicates the need to optimize helix-coil transition
theory parameters. Combining with other NMR techniques, such as CS indices and NOE, this method
will allow us to gain valuable insights on the unfolded state ensemble of MetO-λ and possibly other
protein systems.
POST 09-427
Towards in vivo NMR: Structural Studies Of Prion Fibrils Assembled In Native Environments at
Endogenous Levels
Kendra K. Frederick1, Vladimir K. Michaelis2, Bjorn Corzilius2, Ta-Chung Ong2, Angela C. Jacavone2, Robert
G. Griffin2, Susan Lindquist1, 3, 4
1
Whitehead Institute, Cambridge, Massachusetts, US, 2Chemistry & Francis Bitter Magnet Lab, MIT,
Cambridge, Massachusetts, US, 3Biology, MIT, Cambridge, Massachusetts, US, 4HHMI, Cambridge,
Massachusetts, US
Nearly all biology happens within the boundary of a cell, and intracellular space is highly crowded and
contains a large number of macromolecules with the potential to interact. Yet structural investigations of
biomolecules are typically confined to simplified in vitro systems. Solid-state nuclear magnetic resonance
(NMR) spectroscopy can yield atomic-level information and has no requirement for crystalline samples,
but the ability to investigate low-concentration analytes within complex biological mixtures is in practice
unattainable due to impedingly long acquisition times. Dynamic nuclear polarization (DNP) is able to
dramatically increase the sensitivity of magic angle spinning (MAS) solid-state NMR. Here we apply DNP
MAS NMR to examine an isotopically labeled prion protein, Sup35, assembled into its amyloid form in the
presence of unlabeled cellular lysates. We find that we can specifically observe the protein at endogenous
levels in a complex background in reasonable experimental time, something that would take several
decades to do using traditional MAS NMR. Moreover, we find that while assembly of the amyloid form in
the presence of cell lysates does not perturb the structure of the amyloid core, it results in large changes
in the chemical environment for a portion of the protein that is dynamically disordered in purified
systems. This suggests that the flexible regions of amyloid fibrils may interact with cellular components
that can facilitate assembly into macromolecular structures and inheritance of this protein based element
of inheritance.
POST 09-428
Structural and Ligand Binding Properties of Dimeric Horse Myoglobin
Satoshi Nagao1, Hisao Osuka1, Takuya Yamada1, Takeshi Uni1, Yasuhito Shomura2, 3, Kiyohiro Imai4,
Yoshiki Higuchi2, 3, Shun Hirota1
1
Nara Institute of Science and Technology, Ikoma, Nara, Japan, 2Department of Life Science, Graduate
School of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, Japan, 3RIKEN SPring-8
Center, Sayo-cho, Sayo-gun, Hyogo, Japan,4Department of Frontier Bioscience, Faculty of Bioscience and
Applied Chemistry, Hosei University, Koganei, Tokyo, Japan
266
POSTER ABSTRACTS
Myoglobin (Mb) stores dioxygen in muscles, and is a fundamental model protein widely used
in molecular design. The presence of dimeric Mb has been known for more than forty years, but
its structural and oxygen binding properties remain unknown. From an X-ray crystallographic analysis
at 1.05 Å resolution, we found that dimeric metMb exhibits a domain-swapped structure with two
extended α-helices. Each new long α-helix is formed by the E and F helices and the EF-loop of the
original monomer, and as a result the proximal and distal histidines of the heme originate from
different protomers. The heme orientation in the dimer was in the normal mode as in the monomer, but
regulated faster from the reverse to normal orientation. The dimer possessed the oxygen binding
property, although it exhibited a slightly higher oxygen binding affinity (∼1.4 fold) compared to the
monomer and showed no cooperativity for oxygen binding. The oxygen binding rate constant (kon) of the
dimer ((14.0 ± 0.7) × 106 M−1 s−1) was similar to that of the monomer, whereas the oxygen dissociation
rate constant (koff) of the dimer (8 ± 1 s−1) was smaller than that of the monomer (12 ± 1 s−1). We
attribute the similar kon values to their active site structures being similar, whereas the faster regulation
of the heme orientation and the smaller koff in the dimer are presumably due to the slight change in the
active site structure and/or more rigid structure compared to the monomer. These results show that
domain swapping may be a new tool for protein engineering. Reference Nagao, S., Osuka, H., Yamada,
T., Uni, T., Shomura, Y., Imai, K., Higuchi, Y., and Hirota, S., Dalton Trans. 41, 11378−11385 (2012)
POST 09-429
Study of Interactions of amyloidogenic Regions of Streptococcus mutans adhesin P1 by Nuclear
Magnetic Resonance
Wenxing Tang, L. Jeannine Brady, Joanna R. Long
Unviersity of Florida, Gainesville, Florida, US
Streptococcus mutans is an established etiologic agent of human dental caries, the most common
infectious disease in the world. S. mutans is particularly effective at colonizing hard tissues of the human
oral cavity. Its adherence is mediated by sucrose-dependent and independent mechanisms. In the
absence of sucrose, the extracellular cell wall-associated adhesin P1, also known as Antigen I/II or PAc,
facilitates attachment to the acquired pellicle on teeth. This multifunctional adhesin interacts with the high
molecular weight glycoprotein scavenger receptor gp340, as well as with other oral bacteria and host cell
matrix proteins. Our previous work showed that P1 self-aggregates to form amyloid fibrils and
demonstrates common biophysical properties ascribed to amyloids including uptake of Thioflavin T and
Congo Red (CR) as well as CR-induced birefringence. Amyloid formation is evident within S.
mutans biofilm cultures and known inhibitors of amyloid fibrillization inhibit biofilm formation. P1 has an
unusual structure in which the protein folds back on itself such that an alanine-rich alpha helix intertwines
with a polyproline type II helix to form an extended helical stalk. Beta sheet-rich globular structures
corresponding to the adhesive domains lie on either end of the stalk. Segments containing these globular
regions (A3VP1 and C123) are associated with uptake of amyloidophilic dyes during self-aggregation, and
can also interact with one another. Since P1 was originally identified as a dual antigen comprising antigen
II (C123), and the remainder of the molecule (antigen I), it is likely that interactions between protein
fragments are biologically relevant. The detailed process of amyloid formation is not fully understood.
Here we describe our preliminary studies using high resolution Nuclear Magnetic Resonance to evaluate
the previously crystallized C123 fragment. Shown in Figure 1 is the Transverse Relaxation-Optimized
15
SpectroscopY (TROSY) spectrum of uniformly N labeled C123 in Tris buffer carried out at 25°C with 64
scans and 256 t1 points. By assigning the corresponding residues to these resonance peaks, we will
establish the foundational basis by which to identify key residues and assess conformational changes
associated with intermolecular interactions of C123 with itself during amyloid aggregation, and with
A3VP1. Similar experiments are in process for A3VP1, for which a crystal structure is also available.
267
POSTER ABSTRACTS
POST 09-430
Identification and Characterization of Functional Amyloids in Streptococcus mutans
Richard N. Besingi, L. Jeannine Brady
Oral Biology, University of Florida, Gainesville, Florida, US
Functional amyloid formation is increasingly recognized as a mechanism utilized by microorganisms
(viruses, bacterial, fungi) to facilitate establishment and persistence within their respective environmental
niches. Recently it has been shown that secreted proteins produced by the cariogenic bacterium S.
mutans are capable of amyloid fibrillization. These include but are not limited to adhesin P1 (Ag I/II).
Amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of S.
mutans. Known inhibitors of amyloid fibril formation can inhibit biofilm formation by amyloidogenic
microbes such as S. mutans suggesting a potential target for therapeutic intervention. Although S.
mutans lacking P1 demonstrates residual amyloid forming properties and forms biofilms, a mutant lacking
the transpeptidase enzyme sortase, which covalently links a number of proteins to the peptidoglycan cell
wall, is defective in biofilm formation as well as cell-associated amyloid-like properties. The objective of
this study is to identify additional potential amyloid forming proteins of S. mutans and to determine the
biophysical conditions under which these proteins form amyloids, as well as determine their individual or
combined roles in biofilm formation. To identify amyloid forming proteins in S. mutans, a P1-deficient
mutant strain was grown to stationary-phase in defined minimal media and secreted proteins were
concentrated from spent culture supernatants, followed by fractionation by ion exchange
chromatography. Partially purified protein fractions were tested for binding of the amyloidophilic dyes
Congo Red (CR) and Thioflavin T, and for characteristic birefringent properties following staining with CR
and visualization under a crossed set of polarizing filters. Proteins from fractions that tested positive using
these assays were separated by SDS PAGE, and identified by LC/MS. Proteins identified from
chromatography fractions with amyloid-like properties included WapA, GbpA, GbpB, SMU_2147c and
SMU_63c. Genes encoding these proteins were cloned and recombinant proteins expressed and purified
for confirmation and characterization of individual amyloidogenic properties in vitro. In several instances,
amyloid formation was significantly increased under acidic conditions. This finding is consistent with the
acidogenic and aciduric nature of S. mutans. (Supported by NIH R01DE021789).
POST 09-431
Achieving Selectivity In The Hippo Pathway: An Investigation Of The Inter-Domain Communication
In TEAD Transcription Factors
Priyanka Rauniyar, Sudha Veeraraghavan
Pharmaceutical Sciences, Univ Maryland School Pharmacy, Baltimore, Maryland, US
TEAD is the downstream transcriptional regulator of the Hippo pathway, which plays an important role in
normal organ development, growth, cell proliferation, and apoptosis. Protein co-factors of TEAD include
YAP and TAZ in mammals; defect in TEAD-YAP/TAZ interactions are linked to uncontrolled cell
proliferation, resulting in tumors and cancers. Our group established the first three-dimensional structure
of the DNA-binding TEA domain, which consists of three alpha helices. The N- terminal domain (TEA) is
268
POSTER ABSTRACTS
followed by a 'Linker' domain and then the C-terminal protein-protein interaction domain (CTD). It is not
yet understood how the binding of a protein co-factor at the CTD might influence DNA-binding by the
TEA domain. Here, as part of my undergraduate research internship, I over-expressed and purified the TEA
and CTD and tested for interactions between the domains. Our preliminary results indicate that
interaction between the isolated domains is weak or undetectable suggesting either a role for the linker
domain or for protein cofactors in determining specificity of TEAD proteins. Further biophysical and
biochemical studies on these domains and on full-length TEAD and the DNA complexes are underway.
POST 09-432
Structural Projection of PTMs (SPoP): A Toolkit For Providing Structural And Functional Context
For Sequence-Specific Protein Features
Henry Dewhurst, Matthew P. Torres
While structure is accepted as the key determinant of protein function, the majority of site-specific feature
data (e.g., post-translational modifications (PTMs), mutations) is strictly sequence-linked and lacks
structural context. Here we describe a novel toolkit, Structural Projection of PTMs (SPoP), an application
that bridges this gap by examining protein features in three-dimensional context. SPoP provides a
platform for quantitative and visual analysis of protein features. SPoP is written in Perl, Java, and PyMol
script and is modular in design to allow for rapid integration of additional feature data (e.g. interface
residence and surface accessibility). The toolkit is capable of projecting any feature-set onto a known or
modeled protein structure. The native operating mode employs a composite MySQL database of PTMs
from 12 sources – including user-defined novel PTM sites. Multiple feature-sets may be layered to
generate composite projections. SPoP utilizes either sequential or structural homology for projection of
feature frequencies onto three-dimensional models within PyMol. In addition to visualizations, SPoP’s
analytical core generates raw data on homologous feature detection and conservation of feature sites.
Layering of feature maps allows SPoP to detect overlapping feature-rich regions and identify potential
regulatory sites.
POST 09-433
Investigating the Molecular Basis of Curli Amyloid Inhibition by Protein and Chemical Chaperones
Neha Jain, Margery L. Evans, Matthew R. Chapman
Molecular Cellular and Developmental Biology, University of MIchigan, Ann Arbor, Michigan, US
Proteins can readily adopt stable, ordered aggregates called amyloids. Amyloid formation can be
cytotoxic and is the hallmark of several neurodegenerative diseases. Recently, a new class of amyloids has
been described called ‘functional amyloids’ that are not cytotoxic and instead capitalize on the unique
properties of amyloids to fulfil cellular functions. Functional amyloids are ubiquitous in nature. Curli are
bacterially-produced functional amyloids that are important structural components of bacterial biofilms.
CsgA is the major curli subunit and the assembly of CsgA into extracellular amyloid fibers is a highly
orchestrated process. Therefore, the curli system provides a unique platform to understand the process of
amyloid formation and how it might be inhibited. Here, we present the modulation of curli amyloid
assembly by two classes of molecules: protein and chemical chaperones. We have discovered that
the Escherichia coli periplasmic protein CsgC is an efficient and selective inhibitor of CsgA amyloid
formation. CsgC inhibits CsgA amyloid formation at substoichiometric molar ratios by preventing early
stages of oligomerization in vitro. CsgC also inhibits aggregation of curli amyloid forming proteins from
closely related bacterial species. Furthermore, deletion of CsgC in vivo results in the accumulation of toxic
intracellular CsgA amyloid aggregates. We have also identified and characterized 2-pyridone
peptidomimetic compounds that modulate CsgA amyloid assembly in vivo and in vitro. When added in
269
POSTER ABSTRACTS
combination with another protein chaperone, CsgE, the 2-pyridone compounds act synergistically to
inhibit CsgA amyloid assembly. The 2-pyridone compounds therefore inhibit amyloid assembly through
different mechanisms than protein chaperones. Combined studies of amyloid inhibition by protein and 2pyridone compounds will continue to provide insights into the development of highly specific and
efficient anti-amyloid therapeutics.
POST 09-434
The Effects Of Mutations On The Aggregation Propensity Of Human Prion-Like Domains
Eric D. Ross, Kacy Paul, Sean Cascarina
Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, US
Numerous human proteins contain prion-like domains (PrLDs), defined as domains with similar amino
acid composition to the yeast prion domains. In the past few years, mutations in a number of these PrLDcontaining proteins have been linked to various degenerative diseases. Evidence is emerging that these
PrLDs may play an important role in normal cellular physiology. For example, many of the diseaseassociated PrLDs are found in RNA binding proteins, and result in formation of large cytoplasmic
inclusions. However, formation of naturally-occurring RNA-protein granules is also mediated in part by
PrLDs, suggesting that disease may result from perturbations in normal RNA dynamics. We are using two
proteins, hnRNPA1 and hnRNPA2, as a model system to explore the relationship between PrLD sequence,
aggregation, and pathogenesis. Point mutations in the PrLDs of hnRNPA1 and A2 cause either IBMPFD
(inclusion body myopathy associated with Paget’s disease of the bone and fronto-temporal dementia) or
ALS, and result in formation of large cytoplasmic inclusions. Expression of these proteins causes
mutation-dependent muscle degeneration in Drosophila. In yeast, the mutant prion-like domains are able
to support prion activity, while the wild-type prion-like domains are not. To explore the relationship
between amino acid sequence and aggregation propensity, we have tested approximately 80 hnRNPA1
and A2 mutants in yeast. For the vast majority, the effects of the mutations are accurately predicted by
PAPA (Prion Aggregation Prediction Algorithm), a prediction algorithm designed to predict yeast prion
propensity, suggesting that aggregation propensity can be manipulated in a rational manner. These
mutants will provide a powerful model system to explore the relationship between aggregation
propensity and pathogenesis.
POST 09-435
Competition Or Forced Collaboration? An Unusual Pattern Of Self-Propagating Polymorphism Of
Insulin Amyloid Fibrils Upon Seeding With Mixed Templates.
Wojciech Dzwolak1, 2, Weronika Surmacz-Chwedoruk1, 3
1
Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland, 2Department of
Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland, 3Institute
of Biotechnology and Antibiotics, Warsaw, Poland
B31
B32
Cross-seeding of fibrils of bovine insulin ([BI]) and Lys -Arg human insulin analog ([KR]) induces selfpropagating amyloid variants with infrared features inherited from mother seeds. Here we report an
unexpected outcome of seeding experiments with mixed [BI] and [KR] amyloid templates. When native BI
(or KR) insulin is seeded with mixed equal portions of preformed [BI] and [KR] templates, phenotype of
the resulting daughter fibrils is the same as in the case of purely homologous seeding – i.e. in the absence
of foreign seeds. The selection bias toward homologous daughter amyloid was found to be exceptionally
strong: more than 200-fold excess of heterologous seed was required to imprint its structural phenotype
upon mixed seeding. In the presence of both templates, the fibrillation-promoting and structureimprinting properties of heterologous seeds become uncoupled – heterologous templates still accelerate
fibrillation but have no say in determining phenotype of daughter amyloid (Figure 1). Hence the bias
270
POSTER ABSTRACTS
toward copying the homologous phenotype cannot be explained by kinetic factors only. A hypothetical
explanation is put forward in which: [i] docking of soluble insulin with amyloid tips proceeds through
several steps and [ii] is under thermodynamic control; [iii] free energy of spontaneously formed fibrils is
below energy levels of amyloid variants induced by cross-seeding.
POST 09-436
Nanoscale Organization of Protein Molecules within Amyloids and Prions
Samrat Mukhopadhyay
Department of Biological Sciences and Department of Chemical Sciences, Indian Institute of Science
Education and Research (IISER), Mohali, Mohali, Punjab, India
Amyloids are ordered protein aggregates that are implicated in a variety of debilitating human disorders
such as Alzheimer's, Parkinson's and prion diseases. The transition from a normal functional protein to an
altered (misfolded) form involves a profound conformational change that triggers the aberrant protein
assembly resulting in a wide variety of nanostructures including amyloid oligomers, pores and fibrils. My
laboratory utilizes a diverse array of methodologies to unravel the key molecular events that are crucial in
amyloid formation from a number of proteins. Using Raman spectroscopy in combination with atomic
force microscopy (AFM), we have been able to delineate the key structural transitions during amyloid
formation [1]. The AFM images revealed a progressive morphological transition from spherical oligomers
to nanoscopic annular pores (Fig. 1a), whereas, the Raman data indicated the protein structural changes
during amyloid assembly and pore formation. Recently, we have used the combination of AFM and
Raman to monitor the structural transition of human prion protein into protease-resistant amyloid
oligomers that assemble into ordered fibrils (Fig. 1b) [2]. Additionally, we have adapted a super-resolution
nanophotonic technology that allows us to optically image individual amyloids at the nanoscopic spatial
resolution [3-5]. Due to the optical diffraction-limit, conventional optical microscopy does not allow us to
monitor the nanoscale organization at a high spatial resolution. Therefore, we have utilized near-field
scanning fluorescence microscopy to optically map the amyloid fibrils far beyond the diffraction-limit.
Interrogation of individual fibrils by simultaneously monitoring both nanoscale topography (Fig. 1c) and
fluorescence brightness (Fig. 1d) revealed heterogeneous packing of the cross-β architecture within
amyloids. Our results provide structural underpinnings of diverse amyloid polymorphs that underlie the
strain phenomenon in prion and amyloid biology. [1] M. Bhattacharya, N. Jain, P. Dogra, S. Samai & S.
Mukhopadhyay (2013) J. Phys. Chem. Lett. 4 ,480. [2] V. Dalal & S. Mukhopadhyay (unpublished). [3] V.
Dalal, M. Bhattacharya, D. Narang, P.K. Sharma & S. Mukhopadhyay (2012) J. Phys. Chem. Lett. 3, 1783. [4]
M.Bhattacharya & S. Mukhopadhyay (2014) Nanophotonics, 3, 51. [5] V. Dalal, S. Arya & S. Mukhopadhyay
(2013) in Bionanoimaging: Protein Misfolding & Aggregation (Elsevier; Eds. Y. Lyubchenko & V. Uversky).
POST 09-437
A Temperature Sensitive Parkinsonian Mutation in DJ-1 Enhances Protein Dynamics in a Metaldependent Fashion
Nicole M. Milkovic1, Steven Halouska2, Jonathan Catazaro2, Sara Basiaga3, Robert Powers2, Mark A.
Wilson1
1
Biochemistry and the Redox Biology Center, University of Nebraska-Lincoln, Lincoln, Nebraska,
US, 2Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, US, 3Chemistry Research and
Instrumentation Facility, University of Nebraska-Lincoln, Lincoln, Nebraska, US
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic
neurons in the midbrain. DJ-1 is a small (20 kDa) conserved protein whose absence or inactivation
causes rare forms of inherited parkinsonism. The native function of DJ-1 is cytoprotective, particularly
against oxidative stress and mitochondrial damage. The M26I mutation causes loss of DJ-1 stability and
271
POSTER ABSTRACTS
diminished protein levels in the cell. The crystal structure of M26I DJ-1 is similar to that of wild-type
(WT) DJ-1, showing only minor perturbations near the site of mutation. Thus, it is unclear why this
missense mutation is pathogenic. We have investigated the solution dynamics of WT and M26I DJ-1
using multidimensional NMR spectroscopy. We find that fast timescale dynamics are largely unchanged
between WT and M26I DJ-1; however, longer timescale dynamics are enhanced by M26I DJ-1 at
physiological temperature (37°C), particularly within the hydrophobic core of the protein. Moreover,
M26I DJ-1 shows a marked increase in transition metal-facilitated aggregation in vitro at 37°C,
suggesting that the mutation is pathogenic because it results in a thermally-activated metal-dependent
change in protein dynamics that reduces DJ-1 stability. We propose that the increased conformational
dynamics of M26I DJ-1 lead to protein instability, resulting in a temperature-sensitive and metaldependent loss-of-function phenotype that causes parkinsonism at physiological temperature.
POST 09-438
Analytical Tools in Decision Making of Suitability of Monoclonal Antibodies for Immunodiagnostic
Assays
Kevin R. Rupprecht, Tracey D. Rae, Svetoslava D. Gregory, Na Yang, Janet M. Bergsma, Ryan M. Bonn,
Martin R. Lopez, Panfilo F. Ozaeta, Cheng Zhao, Carol S. Ramsay, Jeffrey R. Fishpaugh
Diagnostic Analytical Chemistry R&D, Abbott Laboratories, Waukegan, Illinois, US
Over the past two decades there has been a rapid and significant rise in the number and types of
analytical methods available for analysis of proteins. These methods are designed to give particular
information about biological molecules such as size, charge characteristics, post transcriptional
modifications and other characteristics. This availability of multiple techniques has led to a realization that
alteration of bioactive molecules can have a profound effect on the activity of those molecules. This is
particularly important when trying to induce cellular systems to produce large amounts of a particular
protein, such as antibodies or antigens for use in immunodiagnostic tests. For example, changes in
growth conditions have been shown to affect glycosylation of monoclonal antibodies. In some cases
these changes to molecular structure may not be easily recognized if one is using methods that only
measure a limited number of protein characteristics, or even using only one method to measure a
particular characteristic. In the immunodiagnostic industry an analytical group is challenged to analyze
large numbers of different antibodies for different antigens in a single year. In this work we detail our
experiences with two particular monoclonal antibodies. In both cases initial analyses did not indicate
extensive variability within or between lots of the monoclonals. There were however subtle clues in the
data that something was amiss with each of these antibodies. By using multiple methods, measuring
multiple characteristics a detailed understanding of the monoclonal structure was obtained for each. In
one case it was discovered that an endogenous light chain was being produced and incorporated into the
monoclonal and in the other case a variable glycosylation of a light chain CDR was found. Both of these
types of changes impact potency and ability of the monoclonal to recognize their respective antigenic
targets. The decisions made that affected recognition of these modifications are discussed.
POST 09-439
Spectroscopic Investigation of the Structural Perturbations of Tau Microtubule Binding Domains at
the Golgi Membrane as Illustrated by Membrane Mimics
Lauren E. Sparks, Larry R. Masterson
Chemistry, Hamline University, St. Paul, Minnesota, US
The demand for understanding certain types of dementia are underscored by limitations for mechanistic
details of the development of Alzheimer’s disease (AD), the only disease in the top ten leading causes of
death that has seen a rise in fatality over the past decade. Hallmarks of AD include plaque formation
272
POSTER ABSTRACTS
created by the amyloid beta protein, and neurofibrillary tangles (NFTs) of the protein, tau. The conserved
microtubule binding domains (MBDs) of tau nucleate the formation of NFTs and have been the subject of
a number of investigations. In vivo studies have shown that NFTs are found in high concentration at the
Golgi membrane (GM) but do not associate with the plasma membrane (PM), suggesting that the GM
may catalyze fibril formation. In order to understand how these regions in tau contribute to the formation
of aggregates, we used peptide fragments corresponding to each of the four conserved repeat segments
in the MBD to investigate their potential interactions with lipid membrane mimics of the GM. To illustrate
that the peptides were sensitive to membrane type, we studied their structural perturbations in the
presence of negatively charged (a mixture of DMPC and DMPG) and uncharged (DMPC only) small
unilamellar vesicles (SUVs) via CD spectroscopy. Introduction of DMPG lipids enhanced the β-sheet
composition of the peptides, a conformational change that is required for NFT formation. Since the GM
surface is more rich in negative charge and potential hydrogen bonding groups than the PM, the
structural changes with PG lipids sheds light on a possible mechanism for protein misfolding events that
lead to NFTs. Further details will be presented on the interaction of the peptides with vesicles based on
lipids that constitute the PM and GM, as well as the influence that these membranes have on the kinetics
of aggregation. These results will help contribute a better understanding for the events that drive tau
aggregation.
273
AUTHOR/SPEAKER INDEX
A
Abe, Ryota POST 10-113
Abedini, Andisheh POST 10-59
Abrol, Ravinder POST 06-367
Adams, Stephen R. POST 03-276
Adams, Paul D. POST 11-140
Adams, Joseph A. POST 03-308
Aebersold, Ruedi POST 06-349
Affandi, Trisiani POST 11-124
Agyekum, Boadi POST 02-229
Ahmad, Shakeel POST 03-295,
POST 12-199
Ahmad, Shadab POST 03-295,
POST 12-199
Ahn, Sae Ryun POST 06-337
Ahn, Kwang H. POST 06-367
Ahn, Natalie G. POST 09-422
Ahn, Natalie POST 03-261
Ahuja, Lalima G. POST 10-67
Allison, Timothy M. POST 06-359
Allison, Brittany POST 12-195
Alon, Assaf POST 12-208
Altshuler, Rachel POST 12-183
Anada, Masahiro POST 06-357
Andersson, Ken POST 12-171
André, Ingemar POST 12-197
Anorma, Chelsea POST 10-116
Arase, Hisashi POST 06-357
Archbold, Julia K. POST 03-233
Arias, Cesar A. POST 10-96
Arreola-Barroso, Rodrigo A. POST
12-178
Arroyo Verástegui, Rossana POST
07-384
Ashraf, Mohd. Tashfeen POST 03295, POST 12-199
Atienza, Carmen POST 12-189
Ausar, Salvador F. POST 10-95
Auton, Matthew POST 10-103
Axe, Jennifer M. POST 03-268
Ayna, Adnan POST 03-243
B
Babbitt, Patricia POST 05-328,
POST 08-400
Baek, Kwang-Hyun POST 04-315
Bah, Alaji POST 12-151
Baker, David POST 03-240
Baker, Tania POST 04-318
Baker, Tania A. POST 04-319
Baker, Dr. David POST 12-214
Baková, Michaela POST 03-284
Balasubramaniam, Deepa POST
10-85
Balch, William POST 10-120
Baldwin, Andrew POST 11-132
Ballard, Keith POST 09-423
Balta, Bulent POST 07-396
Baltz, Morgan R. POST 12-211
Bandak, Diana POST 10-116
Bandi, Swati POST 07-390
Banerjee, Shounak POST 12-183,
POST 12-218
Banister, Sarah POST 03-294
Bao, Ju POST 10-72
Barajas, Jesus POST 03-296
Bardwell, James C. POST 11-126
Barkdoll-Weil, Forest POST 10-90
Barkho, Sulyman POST 03-308
Barragan-Galvez, Juan C. POST
06-336
Bartle, Emily POST 10-89, POST
10-93
Basiaga, Sara POST 09-437
Basore, Danielle A. POST 12-180
Basore, Danielle A. POST 03-287
Batjargal, Solongo POST 03-239
Baugh, Evan POST 03-287
Baxa, Michael C. POST 07-389
Baytshtok, Vladimir POST 04-319
Beatty, Kimberly POST 03-272
Beauregard, Marc POST 03-252,
POST 03-254, POST 03-257, POST
03-259
Beckett, Brian POST 08-405
Beck-Sickinger, Annette G. POST
06-333, POST 10-106
Beeby, Morgan SYMP 05-26
Beelen, Steven POST 07-371
Bell, Anthony POST 12-184
Bell, Ellis POST 03-282, POST 03285, POST 03-290, POST 03-291,
03-311, POST 04-322, POST 04323, POST 10-89, POST 10-90,
POST 10-93
Bell, Jessica POST 03-290, POST
03-291
Bell, Jessica K. POST 02-228
Benesch, Justin POST 11-132
Benítez, Claudia POST 10-98
Benitez Cardoza, Claudia
Guadalupe POST 03-274
Benítez Cardoza, Claudia G. POST
07-384
Benner, W. Henry POST 06-370
Bennett, Eric POST 05-327
Berger, Edward A. POST 12-161
Berger, Edward POST 06-343
Berger, Edward A. POST 10-91
Berghuis, Albert POST 03-250
Bergsma, Janet M. POST 09-438
Berkamp, Sabrina POST 06-360,
POST 06-362
Bernal, Ana POST 11-134
Bernal-Perez, Lina POST 12-209
Berning, Karsten POST 11-128
Besingi, Richard N. POST 09-430
Beuning, Penny J. POST 03-283
Beuron, Fabienne POST 04-321
Bezsonova, Irina POST 08-416
Bhak, Ghibom POST 09-419
Bi, Jing POST 12-159
Bieschke, Jan POST 10-97
Birge, Paige POST 03-294
Biswas, Himadri POST 10-105
Biverstål, Henrik POST 10-86
Bjerneld, Erik POST 08-415
Bjorkman, Pamela SYMP 06-46
Blackledge, Martin POST 10-83
Blackwell, Tim POST 12-222
Blanchette, Craig D. POST 06-368
Blank, Volker POST 12-147, POST
12-148
Blankenship, Robert E. POST 06345
Blois, Tracy POST 06-369
Boassa, Daniela POST 03-276
Bobik, Thomas A. POST 02-225
Bobik, Thomas A. POST 03-310
Boehr, David D. POST 03-248
Boehr, David D. POST 03-268
Bogyo, Matthew SYMP 11-20
Bolduc, Jill M. POST 03-240
Bolon, Dan SYMP 05-25
Bonn, Ryan M. POST 09-438
Bonsor, Daniel A. POST 10-58
Borchers, Christoph H. POST 11126
Borgo, Ben POST 05-329
Botyriute, Akvile POST 09-421
Bourguet, Feliza A. POST 06-368
Bouyain, Samuel E. POST 06-340
Bouyain, Samuel POST 02-229
Bowie, James U. POST 08-409
Bowie, James POST 06-369
Boyd, Courtney POST 03-247
Brady, Jeannine POST 07-387
Brady, L. Jeannine POST 09-429,
POST 09-430
Brajcich, Michelle POST 03-247
Brar, Gloria SYMP 03-01
Brautigam, Chad POST 06-332
Brautigam, Chad A. POST 03-234
Brieba, Luis G. POST 10-98
274
Brieba-Castro, Luis POST 06-336
Brieba de Castro, Luis G. POST 07384
Britton, Robert POST 07-381
Brizendine, Ashley POST 08-399
Broersen, Kerensa POST 10-78
Brooks, Teresa M. POST 10-53
Brooks, Cory L. POST 10-53
Brooks, Cory POST 10-54
Brooks, Shayla A. POST 11-142
Brown, Breann POST 04-318
Brown, Zuben POST 12-185, POST
12-216
Brown, Kyle L. POST 12-222
Bucher, Denis POST 11-129
Budhathoki, Pradeep POST 12-207
Buenavista, Maria Teresa POST
10-104
Buhrman, Jason S. POST 10-114
Bundoc, Virgilio POST 10-91, POST
12-161
Burban, David J. POST 07-392
Bushweller, John POST 10-99
Bustos, Cheene POST 03-310
Butterfoss, Glen POST 03-287,
POST 12-180
Bystroff, Christopher POST 03-287,
12-183, POST 12-218
C
Caaveiro, Jose POST 07-386,
POST 10-87
Caaveiro, Jose M. POST 06-358
Caaveiro, Jose M. POST 10-113
Cabaleiro-Lago, Celia POST 10-68
Caballero, Diego POST 07-393
Cade, Nicholas POST 02-227
Cao, Ping POST 10-59
Capovilla, Alexio POST 06-338
Capraro, Dominique POST 07-392
Carballo-Amador, Manuel A. POST
12-146
Carcamo-Noriega, Edson N. POST
09-425
Carraway, Kermit POST 10-57
Carrillo-Ibarra, Normande POST 03274
Carroll, Candace POST 10-72
Carver, John POST 11-132
Cascarina, Sean POST 09-434
Cascio, Duilio POST 02-230, POST
03-310, POST 11-132
Cash, Jennifer POST 10-55
Catazaro, Jonathan POST 09-437
Cavicchioli, Ricardo POST 11-123
Cerutti, Nichole M. POST 06-338
Cesnekova, Jana POST 04-320
Chacon, Kelly M. POST 06-335
Chadwick, Alexandra POST 03-241
Chakravarty, Suvobrata POST 02226, POST 11-131
Chan, Yan M. POST 03-248
Chan, Michael K. POST 03-278
Chand, Subhash POST 03-267
Chandrashekar, Reena POST 11140
Chánez, Maria E. POST 07-391
Chang, Dennis POST 10-57
Chang, Roger POST 05-331
Chang, Yonggang POST 11-133
Chang, Yu-Chu POST 06-363
Chao, Frank A. POST 08-414
Chapman, Matthew R. POST 09433
Chapman, Matthew SYMP 04-05
Chatani, Eri POST 07-385, POST
10-80
Chattopadhyaya, Rajagopal POST
10-105
Chaudhary, Nidhee POST 03-267
Chaudhary, Akash POST 03-295,
POST 12-199
Chen, Yiliang POST 03-241
Chen, Yao POST 10-69
Chen, Xi POST 12-219
Chen, Liqing POST 06-366
Cheung, Hon-Yeung POST 03-299
Chiang, Chu-Harn POST 07-388
Chica, Roberto A. POST 12-173
Chmielewski, Jean POST 12-144
Chmielewski , Jean POST 10-50
Choi, Angela POST 12-183
Chong-yang, Zhu POST 03-300,
POST 03-303
Choudhury, Shilpa POST 06-351,
POST 08-412
Chowdhury, Chiranjit POST 02-225
Chowdhury, Sudeshna POST 03281
Christ, Daniel POST 08-397
Chuang, Kuo-Hsiang POST 12-193
Chun, Sunny POST 02-225
Chung, Hokyung K. POST 03-242
Church, George POST 05-331
Churchfield, Lewis POST 08-414
Churchman, L. Stirling SYMP 03-02
Clancy, Shonda POST 10-117
Clarke, Jane POST 12-169, SYMP
01-23
Clasman, Jozlyn R. POST 03-263
Colelli, Kathryn POST 03-231
Coleman, Matthew A. POST 10-57
Coleman, Matthew A. POST 06-368
Coleman, Matt POST 06-370
Collier, Miranda POST 11-132
Collyer, Charles A. POST 10-77
Colon, Selene POST 12-222
Corbo, Claudia POST 06-355
Correa-Basurto, Jose POST 03-274
Cortajarena, Aitziber L. POST 12189
Corzilius, Bjorn POST 09-427
Couleaud, Pierre POST 12-189
Cox, Dezerea POST 11-132
Craig, Connor P. POST 03-253
Cravatt, Benjamin F. POST 03-280
Cravatt, Benjamin SYMP 07-13
Crawford, Terry POST 04-316
Crone, Donna E. POST 12-183,
POST 12-218
Crosby, Christine EW II-48
Crowhurst, Karin A. POST 11-142
Crowley, Paula POST 07-387
Crump, Matthew . POST 12-150
Cui, Li POST 03-254
Cukalevski, Risto POST 10-65
Culp, Megan POST 10-112
Curmi, Paul M. POST 11-123
Curnow, Paul POST 06-344, POST
06-346, POST 06-348, POST 12166
D
da Fonseca, Paula POST 04-321,
SYMP 11-19
Daman, Andrew POST 12-191
Das, Bibhuti POST 06-360, POST
06-362
Dattelbaum, Jonathan POST 12167
Davey, James A. POST 12-173
Davis, Ellen POST 10-55
Davis, Joseph H. POST 07-381
Davlieva, Milya POST 10-96
Day , Richard POST 12-200
De Angelis, Anna POST 06-360,
POST 06-362
Deb, Arpan POST 06-366
Deerinck, Thomas J. POST 03-276
Deka, Swapnav POST 06-332
Deka, Ranjit K. POST 03-234
DeLisa, Matthew P. POST 12-153
DeLisa, Matthew P. POST 12-211
Dembinski, Holly E. POST 11-137
Demir, Ozlem POST 10-101
275
Dennis, Edward A. POST 11-129
Derewenda, Zygmunt POST 12-188
Deroo, Stéphanie POST 06-349
Deshayes, Kurt POST 04-316
De Sutter, Delphine POST 05-326
Dewhurst, Henry POST 06-351,
POST 08-412, POST 09-432
Dey, Moul POST 11-131
Dey, Barna POST 10-91
Dickson, Alan J. POST 12-146
Di Lello, Paola POST 04-316
DiMaio, Daniel POST 06-335
Dinler-Doganay, Gizem POST 07396
Dobson, Renwick C. POST 03-236
Dodard-Friedman, Isadore POST
12-148
Dolfe, Lisa POST 10-86
Donald, Bruce R. POST 11-130
Dordick, Jonathan S. POST 12-183,
POST 12-218
Doucet, Nicolas POST 11-139
Dowhan, William SYMP 12-38
Doyle, Shannon POST 11-135
Drobnick, Joy POST 04-316
Drummond, Jake POST 04-316
Duda, David POST 10-69
Duellberg, Christian POST 02-227
Duhr, Stefan POST 10-92
Dvir, Hay POST 10-81
Dzwolak, Wojciech POST 09-435
E
Ebert, Maximilian POST 03-250
Ecroyd, Heath POST 11-132
Eda, Kazuo POST 07-385
Efremov, Rouslan POST 06-349
Eisenberg, David POST 11-132,
POST 12-188
Eisenmesser, Elan Z. POST 10-71
Eitan-Wexler, Maayan POST 08404
Elahi, Montasir POST 08-398
Eller, Chelcie H. POST 10-110
Ellisman, Mark H. POST 03-276
Elpidina, Elena N. POST 03-298
Elpidina, Elena POST 03-251
Engler , David A. POST 06-355
Engler , Anthony C. POST 06-355
Ernst, James POST 04-316
Escalante, Carlos R. POST 10-115
Essel, Francisca POST 02-226,
POST 11-131
Euler, Christian K. POST 12-173
Evangelopoulos , Michael POST
06-355
Evans, Margery L. POST 09-433
Expression group, Structural
Biology POST 04-316
Eyckerman, Sven POST 05-326
F
Fairlie, David P. POST 03-233
Fallis, Lyndsey POST 03-298
Fan, Jin-Yuan POST 02-229
Faraggi, Eshel POST 07-383
Farmer, Rohit POST 12-150
Farrell, Eric POST 11-138
Fekner, Tomasz POST 03-278
Fetrow, Jacquelyn POST 05-328,
08-408
Fetrow, Jacquelyn POST 08-406
Fetrow, Jacquelyn S. POST 08-410
Filippova, Irina Y. POST 03-298
Filippova, Irina POST 03-251
Fischer, Nicholas O. POST 06-368
Fishpaugh, Jeffrey R. POST 09-438
Fleetwood, Filippa POST 12-171
Fleishman, Sarel J. POST 12-208
Fleishman, Sarel POST 12-170
Floyd, Sally POST 05-330
Ford, Nicole POST 10-92
Forman-Kay, Julie POST 12-151,
SYMP 04-08
Forouhar, Farhad POST 03-240
Forsaith, Marc POST 06-348
Frank, Matthias POST 06-370
Fraser, Keith POST 12-183
Frederick, Kendra K. POST 09-427
Freed, Karl F. POST 07-389
Friedler, Assaf POST 10-58
Frohm, Birgitta POST 10-65
Fromme, Petra POST 06-366
Fry, Michelle Y. POST 08-405
Fuchs, Stephen POST 02-224
Fuglebakk, Edvin POST 03-256
Fujii, Satoshi POST 12-210
Fujii, Yuki POST 12-205
Fujimoto, Mayu POST 03-304
Fukakusa, Shunsuke POST 10-107
Fukamizo, Tamo POST 03-244,
POST 03-245
Furuie, Yoshito POST 07-385
Furukawa, Atsushi POST 06-357
Fusa, Shuhei POST 07-380
G
Galuszka, Petr POST 03-284
Gao, Jinlong POST 10-77
Garcia, Jorge D. POST 03-269
Garcia, Ponciano POST 10-98
Garcia, Chris SYMP 12-37
Garcia-Diaz, Miguel POST 03-301
Gardner, Nathan POST 07-372
Garreta, Luis POST 12-183
Gassaway, Brandon POST 12-164
Gemeinhart, Richard A. POST 10114
Gene, Robert POST 10-54
Geng, Xi POST 12-175
Gevaert, Kris POST 05-326
Ghanem, Mustafa POST 10-91
Ghribi, Manel POST 03-252
Gilbert, Erin POST 12-183
Gilmore, Petra E. POST 06-354
Gintner, Lucas POST 12-194
Glasner, Margaret E. POST 08-399
Gleaton, Jeremy POST 12-144
Glukhova, Alisa POST 10-52
Goddard III, William A. POST 06367
Goldman, Adrian POST 10-118
Goldschmidt, Luki POST 12-188
Gonzalez, Teresa POST 12-189
Goodwin, Douglas POST 03-277
Goptar, Irina POST 03-251
Goto, Natalie K. POST 12-213
Goulding, Celia W. POST 08-407
Govinda Remesh, Soumya POST
10-115
Greene, Lesley H. POST 08-402
Gregory, Svetoslava D. POST 09438
Griffin, Michael D. POST 03-236
Griffin, Robert G. POST 09-427
Gross, Richard POST 12-180
Gross, Richard A. POST 03-287
Grove, Tijana POST 12-175
Grove, Tijana Z. POST 12-177
Gruschus, James M. POST 08-418
Gruszka, Dominika T. POST 12-169
Gui, Shanying POST 03-286
Guja, Kip POST 03-301
Gunsel, Umut POST 07-396
Gurrola Acosta, MIsrain E. POST
07-391
Guzman, Carol POST 10-90
H
Hackel, Benjamin POST 12-143
Hackel, Benjamin J. POST 12-182
Haddadian, Esmael POST 07-389
Hadi, Masood Z. POST 06-368
276
Hahn, Klaus SYMP 10-36
Hailey, Kendra POST 10-100
Haimovich, Adrian POST 12-164
Haines, Anthony S. POST 12-150
Hajduczki, Agnes POST 12-161
Halili, Maria A. POST 03-233
Halouska, Steven POST 09-437
Hamakubo, Takao POST 07-386
Hams, Nicole POST 06-365
Handel, Tracy POST 06-353
Handley, Lindsey D. POST 10-119
Hannan, Ross POST 03-297
Hannan, Katherine POST 03-297
Harada, Erisa POST 11-127
Harper, Angela POST 08-401
Harry, David POST 04-322, POST
04-323
Hashimoto, Shunichi POST 06-357
Haugner, John C. POST 08-414
Hau-Riege, Stefan POST 06-370
Havranek, Jim POST 05-329
Hayden, Julia POST 08-408
Hayes, Christopher S. POST 08407
Hayre, N. Robert POST 12-219
He, Wei POST 06-368, POST 10-57
He, Liu POST 03-302
Heald, Rebecca SYMP 08-17
Hébert-Ouellet, Yannick POST 03259
Hecht, Michael SYMP 09-29
Heddle, Jonathan G. POST 12-185
Heddle, Jonathan G. POST 12-216
Hefzi, Hooman POST 05-331
Heim, Erin N. POST 06-335
Heim, Kyle P. POST 07-387
Heine, Holger POST 10-79
Heirbaut, Michelle POST 07-371
Henderson, Paul POST 10-57
Henderson, Rory POST 11-141
Henry, Nicolas POST 06-349
Henry, Ralph POST 11-141
Hermansson, Erik POST 10-86
Hernández-Vázquez, Christian A.
POST 03-307
Herrera, Esperanza POST 03-307
Hess, Sara K. POST 06-350
Hevel, Joan POST 03-286
Heyes, Colin D. POST 11-140
Heyes, Colin POST 11-141
Higuchi, Yoshiki POST 09-428
Hilvert, Donald POST 08-417
Hinkovska-Galcheva, Vania POST
10-52
Hinsen, Konrad POST 03-256
Hirosawa, Narumi POST 03-289
Hirota, Shun POST 09-428
Hluska, Tomáš POST 03-284
Hoang, Quyen POST 10-87
Hoang-Phou, Steven POST 10-57
Hochberg, Georg POST 11-132
Hockla, Alexandra POST 12-217
Holden, Jeffrey K. POST 10-70
Holden, Lauren POST 06-353
Holec, Patrick POST 12-143
Holme, Rebecca L. POST 03-241
Holmes, David POST 02-227
Holton, Thomas POST 12-188
Honda, Shinya POST 12-160
Honda, Takeshi POST 10-107
Hongo, Kunihiro POST 07-380,
POST 07-382
Hoppe, Adam POST 11-131
Hori, Hiroshi POST 07-385
Horiuchi, Masataka POST 10-108
Horiuchi, Rumi POST 10-108
Horng, Jia-Cherng POST 07-388
Horowitz, Scott POST 11-126
Hoskins, Joel POST 11-135
Houk, Kendall N. POST 02-225
House, Colin POST 03-297
Houstek, Josef POST 04-320
Hsu, Chih-An POST 03-237
Hu, Jiancheng POST 10-67
Huang, Yao-ming POST 12-174,
POST 12-183
Huang, Yao-Ming POST 12-218
Huang, Tai-huang POST 04-324
Huber, Thaddaus R. POST 12-221
Hubin, Ellen POST 10-78
Hudson, Billy POST 12-222
Hughes, Roy POST 09-426
Huh, Ian POST 10-54
Hui, Elliot E. POST 03-273
Hunter, Mark POST 06-370
Huo, Yunwen POST 03-242
Hussain, Rohanah POST 10-104
Hyde, Alaina POST 10-93
I
Ichihashi, Norikazu POST 08-411,
POST 12-162
Ieong, Pek POST 10-101
Iida, Tetsuya POST 10-107
Iizuka, Ippei POST 03-304
Ikeda, Masashi POST 07-380
Imai, Kiyohiro POST 09-428
Imamoglu, Rahmi POST 07-396
Inaba, Kenji POST 03-233
Inaka, Koji POST 03-304
Iosub-Amir, Anat POST 10-58
Irbäck, Anders POST 09-424
Isaacs, Farren POST 12-164
Ishizuka-Katsura, Yoshiko POST
06-356
Issaian, Aaron V. POST 11-124
Iwamoto, Aikichi POST 06-341
Iwasaki, Kenji POST 12-185, POST
12-216
J
Jacavone, Angela C. POST 09-427
Jackman, Brianna M. POST 03-263
Jackson, Sophie E. POST 07-375
Jackson, Paul J. POST 06-368
Jacobs, Conor POST 03-242
Jacobsen, Michael T. POST 12-156
Jäger, Linda POST 08-417
Jaikaran, Anna S. POST 12-151
Jain, Nikhil POST 07-381
Jain, Nitin POST 11-134
Jain, Neha POST 09-433
Jamin, Marc POST 10-83
Jarvik, Nick POST 12-212
Jefferson, Robert POST 06-369
Jeng, Wen-Yih POST 03-237
Jen-Jacobson, Linda POST 03-270
Jennings, Patricia A. POST 03-308,
POST 10-100, POST 10-121
Jennings, Patricia POST 07-392
Jensen, Malene R. POST 10-83
Jez, Joseph M. POST 03-260
Jiang, Zhiping POST 10-75
Jo, Hyunil POST 12-202
Joachimiak, Andrzej POST 10-117
Johansson, Jan POST 10-86
Johansson, Johan POST 08-415
Johnson, Parker M. POST 08-407
Johnson, Lucas POST 12-194
Johnson, Troy POST 10-96
Johnson, Colin P. POST 06-365
Johnson, William POST 06-366
Jomaa, Admad POST 07-381
Jones, Krysten A. POST 03-273
Jones, Victoria POST 12-183
Jones, Rachel POST 03-282
Jones, Bryan E. POST 10-100
Jònsson, Sigurdur Æ. POST 09-424
Jorda, Julien POST 03-310
Jugnarain, Vinesh POST 06-338
Jun, Zhang POST 03-258, POST
03-300, POST 03-303, POST 10-94
June, Cynthia M. POST 03-263
277
K
Kadumoori, Rajashekar V. POST
12-201
Kaempfer, Raymond POST 08-404
Kailasan, Shweta POST 07-387
Kakita, Kosuke POST 06-357
Kalastavadi, Tejas POST 04-318
Kamali-Moghaddam, Masood POST
10-109
Kamer, Kimberli J. POST 03-232
Kamiya, Narutoshi POST 12-163
Kang, Soosung POST 10-70
Kannan, Meenakshi B. POST 12147
Kantserova, Nadezda P. POST 1061, POST 10-64
Kao, Tang-Chun POST 07-388
Karamitros, Christos S. POST 1066
Karonicolas, John POST 12-183
Karsai, Arpad POST 12-219
Kast, Peter POST 08-417
Kategaya, Lorna POST 04-316
Kato, Masato POST 09-420
Kaur, Nameet POST 03-267
Kawahara, Kazuki POST 10-107
Kawata, Yasushi POST 07-380,
POST 07-382
Kay, Michael S. POST 12-156
Kay, Brian K. POST 06-368
Kazuta, Yasuaki POST 08-411,
POST 12-162, POST 12-210
Kehr, Andrew D. POST 03-270
Keillor, Jeffrey W. POST 12-213
Kelly, Robert J. POST 10-52
Kendall, Debra E. POST 06-367
Kendrick, Agnieszka A. POST 1071
Kerckhove, Clara POST 03-291
Kerstetter, Nicole E. POST 03-268
Kesavardhana, Sannula POST 12165
Kessans, Sarah A. POST 03-236
Khare, Sagar D. POST 03-240
Khatri, Vinay POST 03-259
Khersonsky, Olga POST 12-170
Khowala, Suman POST 03-281
Kim, So-Ra POST 04-315
Kim, Jin-Ok POST 04-315
Kim, Hyun-Sook POST 10-60
Kim, Seungkyung POST 03-255
Kim, Inhae POST 08-409
Kim, Sanguk POST 08-409
Kim, SoHo POST 03-290
Kimoto, Hisashi POST 03-244
Kines, Kelsey POST 04-322, POST
04-323
Kingdom, Grace POST 03-311
Kleanthous, Colin SYMP 02-40
Klein, Mark A. POST 12-215
Klinger, Neil V. POST 03-266
Kloczkowski, Andrzej POST 07-383
Kluber, Alexander J. POST 12-219
Knight, Michael POST 06-344,
POST 06-346, POST 12-166
Knight, Mary J. POST 02-230
Knowles, Tuomas POST 10-65
Knox, Curtis POST 12-196
Knutson, Stacy POST 05-328,
POST 08-400
Kobayashi, Yuji POST 10-107
Kodama, Tatsuhiko POST 07-386
Koide, Akiko POST 12-176
Koide, Shohei POST 12-176
Köllisch, Gabriele POST 10-79
Kolodziejczyk, Robert POST 10-118
Komives, Elizabeth POST 10-85,
POST 11-137
Komives, Elizabeth A. POST 03288
Komives, Elizabeth A. POST 10119
Konkle, Mary POST 03-262, POST
03-294
Konrad, Manfred W. POST 10-66
Konuma, Tsuyoshi POST 11-127
Kornev, Alexandr P. POST 10-67
Kosel, David POST 06-333
Kovacich, Chelsea POST 12-196
Krammer, Eva-Maria POST 06-349
Kratzke, Marian POST 12-215
Krendel, Mira POST 12-159
Krishnan, Krish POST 11-136
Krishnan, Yamuna SYMP 07-12
Krumm, Stefanie A. POST 03-242
Kruziki, Max POST 12-143
Krylov, Vyacheslav V. POST 10-64
Kudo, Shota POST 07-386
Kumar, Anil POST 12-151
Kumar, Aditya A. POST 12-165
Kumar, Kiran POST 08-406
Kumar, Suresh POST 11-141
Kumaran, Jyothi POST 10-54
Kumeta, Msahiro POST 07-377
Kung, Chia-Hsiuan C. POST 04-324
Kuntimaddi, Aravinda POST 10-99
Kuroda, Yutaka POST 08-398
Kuroki, Kimiko POST 06-357
Kurpiewski, Michael R. POST 03270
Kusaoke, Hideo POST 03-244
Kwok, Michael POST 04-316
Kwong, Peter SYMP 06-44
Arroyo, Rossana POST 10-98
Cox, Daniel L. POST 12-219
DeGrado, William F. POST 12-202
Geli Fernandez-Penaflor, Maria
Isabel SYMP 10-32
Ladbury, John POST 10-96
L
Laffoon, Megan POST 03-262
Laganowsky, Arthur POST 11-132
Laganowsky, Art POST 06-359
Lai, Yen-Ting POST 12-155
Lam, Stephanie S. POST 03-232
Lam, Kit S. POST 06-368
Lamberson, Colleen POST 12-183
Lamoureux, Guillaume POST 03250
Landau, Mark SYMP 07-10
Langley, David POST 08-397
Latham, Michael P. POST 09-422
Laurence, Ted A. POST 06-368
Lawrence, Kenneth F. POST 02228
Lazic, Ana EW II-48, POST 10-92
Lee, Cheng-Chung POST 03-237
Lee, Thomas POST 09-422
Lee, Soon Goo POST 03-260
Lee, Ho-Jin POST 10-72
Lee, Heewon POST 10-74
Lee, Jennifer C. POST 10-75
Lee, Jennifer C. POST 06-350
Lee, Marianne M. POST 03-278
Leettola, Catherine N. POST 02230
Lee†, Kyunglim POST 10-74
Leimer, Tobias POST 06-333
Leket-Mor, Tsafrir POST 06-366
Lenobel, René POST 03-284
Leonard, David A. POST 03-263,
POST 03-264
Leonard, Paul POST 10-96
Lermyte, Frederik POST 07-371
Leung, Isabel POST 12-212
Leuthaeuser, Janelle POST 05-328,
08-406
Leuthaeuser, Janelle POST 08-408
Leuthauser, Janelle B. POST 08410
Lev-Ram, Varda POST 03-276
Lew, Scott POST 03-240
Lewis, Nathan E. POST 05-331
278
Leyrat, Cédric POST 10-83
Li, Linqiu POST 03-299
Li, Wenzhou POST 09-423
Li, Nan POST 10-77
Li, Kan POST 09-426
Li, David POST 03-273
Li, Chun POST 03-292
Li, Xiao-dan POST 06-370
Liao, Xiaoli POST 12-179
Liddle, Jen POST 03-261
Lim, Nicole C. POST 07-375
Lin, Michael Z. POST 03-242
Lin, Tao POST 02-226, POST 11131
Lin, Pen-Jen POST 04-325
Lin, Eugene POST 06-361
Lin, Qingsong POST 06-364
Lindahl, Fredrik POST 03-233
Lindquist, Susan POST 09-427
Linse, Sara POST 10-65, POST 1068
Lipper, Colin H. POST 10-121
Liu, Wei Z. POST 03-234
Liu, Chang POST 12-149
Liu, Wei-Chun POST 03-237
Liu, Cong POST 11-132
Liu, Li POST 10-91
Liu, Gang-yu POST 12-219
LiWang, Andy POST 11-133
Lobner, Elisabeth POST 12-203
Löf, Liza POST 10-109
Löfblom, John POST 12-171
Loh, Stewart N. POST 12-159
Lokensgard, Melissa E. POST 12181
Lomax, Jo E. POST 10-110
Long, Joanna R. POST 09-429
Lopes, Nicholas POST 11-134
Lopez, Mariana POST 08-399
Lopez, Javier POST 12-189
Lopez, Martin R. POST 09-438
Love, John J. POST 03-313, POST
03-314, POST 12-181
Low, David A. POST 08-407
Low, David SYMP 02-42
Lu, Jia POST 03-278
Luisillo-Quiñones, Ericka Fabiola
POST 03-307
Luo, Ray POST 03-296
Lu-yu, Zhang POST 10-94
Ly, Cuong POST 04-316
Lysenko, Liudmila A. POST 10-61,
POST 10-64
M
Mabbutt, Bridget C. POST 12-186
MacInnes, Katherine A. POST 06348
Madde, Pranathi POST 10-103
Maenaka, Katsumi POST 06-357
Maeng, Jeehye POST 10-74
Maestre-Reyna, Manuel POST 03237
Maffitt, Mark POST 05-330
Majumdar, Sangita POST 03-281
Majumder, Erica L. POST 06-345
Majumder, Rajib POST 03-281
Makepeace, Karl A. POST 11-126
Makhatadze, George I. POST 03287
Malay, Ali A. POST 12-185
Malay, Ali D. POST 12-216
Malisauskas, Ricardas POST 09421
Mallela, Krishna POST 07-390
Mallon, Ann-Marie POST 10-104
Mamat, Uwe POST 12-196
Manea, Francesca POST 12-186
Mann, Stephen POST 12-166
Marada, Suresh POST 10-72
Marana, Sandro R. POST 03-279
Marcos, Enrique POST 12-214
Marcotte, Ed SYMP 03-04
Marean-Reardon, Carrie POST 12158
Margulies, David POST 03-305
Marieni, Michelle POST 03-231
Marion, James D. POST 03-288
Marston, Jez L. POST 06-335
Marszalek, Piotr POST 07-394,
POST 07-395
Martell, Jeffrey D. POST 03-232
Martin, Jennifer L. POST 03-233
Martin, Esther POST 07-371
Martin, Jeffrey W. POST 11-130
Martin, Rachel W. POST 10-116
Martínez-François, Juan Ramón
POST 03-249
Masaki, Mika POST 03-304
Mascola, John R. POST 12-165
Masson, Laura POST 12-167
Masterson, Larry R. POST 03-271,
POST 09-439
Masterson, Larry POST 10-112
Masuda, Yuuki POST 10-80
Masuda, Akemi POST 12-192
Matsuda, Zene POST 06-341
Matsuura, Tomoaki POST 12-210
Matsuura, Tadashi POST 07-386
Mattern, Andreas POST 10-106
Maurer, Till POST 04-316
Mays, Jacqunae POST 03-285
McAteer, Kathleen POST 12-158
McCammon, J. Andrew POST 11129
McEvoy, Megan M. POST 11-124
McFarland, Benjamin J. POST 12191
McGuffin, Liam James POST 10104
McKenna, Robert POST 07-387
McKinley, Sean W. POST 02-228
McKnight, Steven POST 09-420
McMillan, Andrew POST 08-399
McNamara, Dan E. POST 03-310
Mead, David POST 05-330, POST
12-196
Meddeb, Fatma POST 03-254
Meddeb-Mouelhi, Fatma POST 03252, POST 03-257, POST 03-259
Mehl, Ryan POST 06-365
Meiler, Jens POST 12-195
Meisl, Georg POST 10-65
Mejias, Sara H. POST 12-189
Mendoza-Cozatl, David G. POST
03-269
Meng, Fanling POST 10-59
Menze, Michael POST 03-262,
POST 03-294
Mercedes-Camacho, Ana POST 12177
Metz, Carlos POST 10-93
Meyer, Matthew R. POST 06-347,
POST 06-352
Meyer, Matthew R. POST 06-354
Michaelis, Vladimir K. POST 09-427
Middleton, Chris T. POST 10-59
Mikula, Kornelia M. POST 10-118
Milkovic, Nicole M. POST 09-437
Millan-Pacheco, Cesar POST 07374
Miller, Liz SYMP 01-21
Mills, Kenneth POST 03-231
Mills, Jeremy POST 03-240
Minardi, Luke POST 12-194
Ming-ming, Lin POST 03-238,
03-303
Mir, Stephan POST 10-85
Mirasol Melendez, Elibeth POST
03-274
Mirasol Meléndez, Elibeth POST
07-384
Mirzaee, Nima POST 12-219
Mitchell, Joshua M. POST 03-264
Mitchell, Joshua M. POST 03-293
279
Mitternacht, Simon POST 09-424
Mittler, Ron POST 10-121
Mizobata, Tomohiro POST 07-380,
POST 07-382
Mizuta, Toshifumi POST 07-382
Mizutani, Kenji POST 06-356
Moisan, Jessica K. POST 03-257
Molinaro, Roberto POST 06-355
Mongeon, Rebecca POST 03-249
Montelongo, David M. POST 10116
Moody, Peter POST 03-243
Moon-Tasson, Laurie POST 10-103
Mootha, Vamsi K. POST 03-232
Mootz, Henning D. POST 03-246
Morales, Yalemi POST 03-286
Morante, Koldo POST 10-113
Morelli, Aleardo POST 08-414
Moreno-Sánchez, Rafael POST 03269
Morgan, Richard POST 10-91
Morikawa, Yasushi POST 03-304
Moriwaki, Yoshitaka POST 10-113
Mörl, Karin POST 06-333
Morrill, Summer M. POST 02-224
Morris, Edward POST 04-321
Morris, Kyla POST 11-140
Morse, Robert P. POST 08-407
Mosaheb, Mohammad POST 02224
Mou, Yun POST 12-206
Mouchlis, Varnavas POST 11-129
Moulick, Roumita POST 07-378
Movahedin, Reza POST 10-53
Mueller, Karl T. POST 06-339
Mukhopadhyay, Samrat POST 09436
Mulligan, Vikram K. POST 03-240
Mundorff, Emily POST 12-190
Mura, Cameron POST 07-379
Murata, Takeshi POST 06-356
Murray, Jeremy POST 04-316
N
Nagai, Kazuma POST 12-187
Nagao, Satoshi POST 09-428
Nagatoishi, Satoru POST 07-386
Naik, Mandar T. POST 04-324
Najnin, Tahria POST 11-123
Nakamura, Haruki POST 12-163
Nakamura, Kazuo T. POST 03-304
Nakamura, Shota POST 10-107
Nakane, Shuhei POST 06-341
Naldrett, Michael J. POST 04-317
Nam, Hyun Jun POST 08-409
Nasir, Irem POST 10-68
Naumann, Todd A. POST 04-317
Ndontsa, Elizabeth POST 03-277
Ndubaku, Chudi POST 04-316
Nechushtai, Rachel POST 10-121
Nemova, Nina N. POST 10-61,
POST 10-64
Nepal , Manish POST 10-50
Newberry, Robert W. POST 07-373
Ngo, John T. POST 03-276
Ngo, Alice POST 12-219
Ngu, Lisa POST 03-283
Nguyen, Nhung T. POST 11-139
Nguyen, Long POST 12-184
Nicastri, Michael POST 03-231
Nikolaienko, Roman M. POST 06340
Nishida, Takamasa POST 07-385
Nishiyama, Kotaro POST 12-162
Nishiyama, Hiroto POST 12-187
Nitzel, Damon POST 03-286
Njuma, Olive J. POST 03-277
Noel, Joseph P. SYMP 05-27
Noguchi, Keiichi POST 08-398
Nomura, Takao POST 06-357
Nomura, Wataru POST 12-192
Nonaka, Takamasa POST 03-304
Norgard, Michael V. POST 03-234
North, Rachel A. POST 03-236
Numata, Tomoyuki POST 03-245
Nutt, David POST 10-104
O
O'Hara, Danielle N. POST 03-253
O'Hern, Corey S. POST 07-393
O'Neill, Heather POST 09-423
O'Rourke, Kathleen F. POST 03268
Oas, Terrence G. POST 09-426,
POST 11-130
Ochiai, Masanori POST 10-108
Oda, Takashi POST 11-127
Odokonyero, Denis POST 08-399
Ogasawara, Wataru POST 03-304
Ogden, Stacey POST 10-72
Ohkubo, Tadayasu POST 10-107
Ohnuma, Takayuki POST 03-244,
POST 03-245
Ohta, Kazunori POST 03-304
Okada, HIrofumi POST 03-304
Oki, Hiroya POST 10-107
Olajuyigbe, Folasade M. POST 1056
Oliveir, Felipe POST 10-109
Ollis, Anne A. POST 12-153
Omolu, Abbie POST 12-200
Ondrechen, Mary Jo POST 03-283
Ong, Ta-Chung POST 09-427
Onuchic, José N. POST 10-121
Oosaka, Fumina POST 06-357
Opella, Stanley J. POST 06-360,
POST 06-362
Opella, Stanley POST 06-361
Opgenorth, Paul POST 12-204
Oppert, Brenda POST 03-251,
POST 03-298
Ortega, Joaquin POST 07-381
Ortega, Jaime POST 10-98
Ortiz-Navarrete, Vianney POST 06336
Ose, Toyoyuki POST 06-357
Oshiro, Satoshi POST 12-160
Osuka, Hisao POST 09-428
Ozaeta, Panfilo F. POST 09-438
Ozaki, Makoto POST 12-187
P
Paddock, Mark L. POST 10-121
Padmanarayana, Murugesh POST
06-365
Pai, Emil F. SYMP 10-35
Paik, Seung Ryeoul POST 09-419
Palei, Shubhendu POST 03-246
Palida, Sakina F. POST 03-276
Palmer, James POST 08-403
Palsson, Bernhard POST 05-331
Pámanes-Carrasco, Gerardo A.
POST 03-307
Panda, Dulal POST 10-73
Panecka, Joanna POST 07-379
Pardi, Arthur POST 09-422
Paredes, Diana I. POST 12-218
Parish, Carol POST 10-89
Park, Chiwook POST 07-372
Park, Tai Hyun POST 06-337
Park, Jiyong POST 02-225
Park, Hyejin POST 03-290
Park, Sang Ho POST 06-360
Park, Sang Ho POST 06-362
Parker, Rachael POST 12-177
Parnell, Jonathan POST 10-85
Parodi, Alessandro POST 06-355
Pastan, Ira POST 10-51
Pastor, Nina POST 07-374
Pastor, Rich POST 04-316
Patel, Bhavik POST 10-91
Paul, Kacy POST 09-434
Peacock, Riley POST 03-247
Pedchenko, Vadim POST 12-222
Pedrini, Bill POST 06-370
280
Pelletier, Joelle POST 03-250
Pentelute, Bradley POST 12-179
Peralta, Maria D.R. POST 12-219
Pernstich, Christian POST 06-348
Perriman, Adam POST 12-166
Petersson, E. James POST 03-239
Petrotchenko, Evgeniy V. POST 11126
Petti, Lisa M. POST 06-335
Pielak, Gary J. POST 03-312
Pierce, Levi C. POST 03-308
Pita, Liz EW I-47
Pitchai, Ganesha p. POST 10-88
Pitman, Derek J. POST 12-174
Pitman, Derek POST 12-183
Pitman, Derek J. POST 12-188
Plemper, Richard K. POST 03-242
Plesner, Annette POST 10-59
Polikarpov, Igor POST 03-279
Pooe, Ofentse J. POST 10-79
Poortinga, Gretchen POST 03-297
Portnoff, Alyse D. POST 12-211
Potts, Jennifer R. POST 12-169
Poulos, Thomas L. POST 10-70
Powers, Rachel A. POST 03-263,
POST 03-265
Powers, Rachel A. POST 03-266
Powers, Robert POST 09-437
Powers, Rachel POST 03-293
Pozzi, Ambra POST 12-222
Prashek, Jennifer POST 03-255
Prescher, Jennifer A. POST 03-273
Presto, Jenny POST 10-86
Prevost, Martine POST 06-349
Price, Neil P. POST 04-317
Price, Owen POST 03-286
Price, Jeffrey POST 02-229
Prieto, Judith H. POST 10-102
Pritchard, Kirkwood A. POST 03241
Prokopenko, Khristina N. POST 1061
Przybyla, David POST 12-144
Pyun, Haejun POST 10-74
Q
Qi, Yang POST 11-130
Qian-ying, Zhang POST 03-238,
POST 03-258, POST 03-303
Qin, Lingyun POST 10-69
Qin, Ling POST 06-353
Quan, Shu POST 11-126
Quan, Wen POST 03-300, POST
03-303, POST 10-94
R
Rabideau, Amy POST 12-179
Radisky, Evette S. POST 12-217
Radoicic, Jasmina POST 06-360,
POST 06-362
Rae, Tracey D. POST 09-438
Rahman, Nausheen POST 10-95
Raines, Ronald T. POST 07-373,
POST 10-110
Raleigh, Daniel P. POST 10-59
Raman, Bakthisaran POST 10-76
Rämisch, Sebastian POST 12-197
Ramsay, Carol S. POST 09-438
Rana, Rajashree POST 10-72
Ranscht, Barbara POST 06-333
Rao, Gururaj A. POST 06-347
Rao, Ch M. POST 10-76
Rao, Gururaj POST 06-352
Rao, Aragula G. POST 06-354
Rapaport, Hanna POST 12-152
Rape, Michael SYMP 11-18
Rappsilber, Juri SYMP 03-03
Rasche, Madeline E. POST 03-310
Ratcliffe, Sarah POST 06-344,
POST 06-346, POST 12-166
Rathore, Ujjwal POST 12-165
Ratnayake, Punsisi Upeka POST
06-342
Rauniyar, Priyanka POST 09-431
Raussens, Vincent POST 06-349,
POST 10-78
Ray, Shashikant POST 10-73
Rayahin, Jamie E. POST 10-114
Reading, Eamonn POST 06-359
Reardon, Patrick POST 12-158
Reardon, Patrick N. POST 06-339
Reck-Petersen, Sam SYMP 10-34
Rees, Douglas SYMP 12-39
Regan, Lynne POST 07-393, POST
12-157, POST 12-164
Regan, Lynne J. POST 12-200
Reid, Korey M. POST 11-136
Reingewertz, Tali H. POST 10-58
Reinke, Aaron POST 05-327
Reitter, Julie N. POST 03-231
Rendakov, Nikolay L. POST 10-61
Reuter, Nathalie POST 03-256
Reyes-Lopez, Cesar Augusto
Sandino POST 03-274
Rinehart, Jesse POST 12-164
Ripa, Perry POST 12-223
Roberts, Sue A. POST 11-124
Robinson, Carol POST 11-132
Robinson, Carol V. POST 06-359
Rogers, William E. POST 10-62
Rogers, Zachary POST 06-368
Rojo, Arturo POST 10-98
Romo-Astorga, Zaira J. POST 03307
Roos, Hakan POST 08-415
Roppongi, Saori POST 03-304
Roque, Cristopher POST 10-95
Rosario, Rosa POST 10-59
Rosas Trigueros, Jorge L. POST
07-384
Rosenberg, Steven A. POST 10-91
Rosenman, David POST 12-183
Ross, Eric D. POST 09-434
Ross, Gary POST 12-223
Rossjohn, Jamie SYMP 06-45
Rouet, Romain POST 08-397
Rouge, Lionel POST 04-316
Roy, Craig SYMP 02-41
Royappa, Grace POST 10-72
Rubert-Perez, Charles M. POST 12144
Ruigrok, Rob POST 10-83
Rule, Gordon S. POST 03-270
Rupprecht, Kevin R. POST 09-438
Rusek, Marta POST 10-111
Ryu, Youngha POST 12-207, POST
12-209
Š
Šebela, Marek POST 03-284
S
Saab-Rincón, Gloria POST 09-425,
POST 12-178
Saha, Piyali POST 12-165
Sahoo, Daisy POST 03-241
Saibil, Helen SYMP 04-07
Saitoh, Takashi POST 06-357
Sakamoto, Yasumitsu POST 03304
Sakamoto, Jiro POST 06-357
Sakamoto, Takeshi POST 03-289
Sakamoto, Yasushi POST 03-289
Saldana, Matthew POST 10-57
Salvatore, Francesco POST 06-355
Sampson, Nicole POST 03-301
Samyn, Noortje POST 05-326
Sanagavarapu, Kalyani POST 1068
Sandal, Priyanka POST 06-354
Sandros, Marinella POST 10-63
Sangani, Sahil POST 03-270
Sankar, Revathi POST 10-84
Sarroukh, Rabia POST 10-78
Sato, Mamoru POST 11-127
Sauer, Robert T. POST 04-319
281
Sawaya, Michael POST 11-132
Sawyer, Nicholas POST 12-164
Saylor, Benjamin D. POST 03-314
Scaria , Shilpa POST 06-355
Scharadin, Tiffany POST 10-57
Schaub, Andrew J. POST 03-296
Schenkelberg, Christian D. POST
12-174
Scheraga, Harold A. POST 07-376
Schertler, Gebhard POST 06-370
Schiffer, Jamie POST 10-85
Schloss, Ashley POST 12-157
Schloss, Ashley C. POST 12-200
Schmidt, Ann Marie POST 10-59
Scholl, Zackary N. POST 07-394,
POST 07-395
Schulman, Brenda POST 10-69
Scott, Caitlin E. POST 06-367
Scott, Brandon POST 11-131
Sebesta, Jacob POST 12-221
Seedorff, Jennifer POST 06-343
Seelig, Burckhard POST 08-414
Seelig, Burckhard SYMP 05-24
Seetharaman, Jayaraman POST
03-240
Segelke, Brent W. POST 06-370
Sellmyer, Mark A. POST 03-273
Semashko, Tatiana A. POST 03298
Sen, Saurabh POST 05-330, POST
12-196
Senapati, Sanjib POST 10-84
Senior, Laura POST 06-344, POST
06-346, POST 06-348, POST 12166
Serpell, Louise C. POST 10-78
Serrano, Crystal POST 12-184
Sezerman, Osman U. POST 12-168
Shah, Shweta POST 06-347, POST
06-352
Sham, Yuk POST 12-215
Shamoo, Yousif POST 10-96
Sharikova, Valeriya F. POST 03298
Sharikova, Valeriya F. POST 03251
Shastry, Shankar POST 11-135
Shaw, Andrey S. POST 10-67
Shayman, James A. POST 10-52
Shea, Gabrielle B. POST 08-410
Shen, Wei POST 11-128
Sheung, Anthony POST 10-95
Shi, Zhengshuang POST 03-302
Shi, Yiwen POST 10-96
Shih, Hsiu-Ming POST 04-324
Shimba, Noriko POST 12-163
Shinya, Shoko POST 03-244
Shirke, Abhijit POST 12-180
Shirke, Abhijit N. POST 03-287
Shirouzu, Mikako POST 06-356
Shlaifer, Irina POST 03-235
Shoichet, Brian SYMP 07-11
Shomura, Yasuhito POST 09-428
Shonhai, Addmore POST 10-79
Shyur, Lie-Fen POST 03-237
Siddiqui, Khawar S. POST 11-123
Sidhu, Sachdev POST 12-212
Siegel, Justin B. POST 12-172
Silverman, Richard B. POST 10-70
Simonyan, Lusine POST 11-142
Singh, Surinder POST 07-390
Singh, Rajiv R.P. POST 12-219
Singh, Jay POST 10-120
Sinha, Kaustubh POST 03-270
Sljoka, Adnan POST 06-334
Smirnova, Yulia POST 03-251
Smirnovas, Vytautas POST 09-421
Smith, Amber M. POST 11-125
Smith, Austin E. POST 03-312
Smith , Janet L. POST 11-125
Snow, Christopher POST 12-194,
POST 12-221
Sobott, Frank POST 07-371
Solamo, Felix EW III-49
Song, Fei POST 10-59
Sori, Nardos POST 08-402
Sosnick, Tobin R. POST 07-389
Sot, Begoña POST 12-189
Souvignier, Chad POST 12-196
Sparks, Lauren POST 03-271
Sparks, Lauren E. POST 09-439
Squier, Thomas POST 12-158
Srinivas, Raja R. POST 12-203
Srinivas, Dustin POST 06-366
Ståhl, Stefan POST 12-171
Steinmetz, Eric POST 05-330,
POST 12-196
Stengel, Florian POST 06-349
Stephens, Erin A. POST 12-211
Stiburek, Lukas POST 04-320
Stock, Daniela POST 08-397
Stoddard, Barry L. POST 03-240
Strelkov, Sergei POST 07-371
Strmiskova, Miroslava POST 12213
Stroud, James POST 11-132
Strutz, Wyatt POST 10-92
Su, Zhengding POST 10-69
Su, An POST 03-287
Sudou, Yukio POST 07-386
Sugase, Kenji POST 11-127, POST
12-154
Sulkowska, Joanna POST 07-392
Sultan, Abdullah POST 10-76
Sunami, Takeshi POST 08-411,
POST 12-210
Sundberg, Eric J. POST 10-58
Suon, Peter POST 03-313
Surmacz-Chwedoruk, Weronika
POST 09-435
Surrey, Thomas POST 02-227,
SYMP 08-16
Suzuki, Hironori POST 03-236
Suzuki, Yoshiyuki POST 03-304
Suzuki, Kano POST 06-356
Svensson, Erika POST 08-415
T
Taira, Toki POST 03-245
Takahashi, Satoshi SYMP 01-22
Takeyasu, Kunio POST 07-377
Tallman, Katie POST 03-272
Tamaki, Fabio K. POST 03-279
Tamamura, Hirokazu POST 12-192
Tamang, Sanjeeta POST 03-281
Tanaka, Hiroaki POST 03-304
Tanaka, Nobutada POST 03-304
Tanaka, Koji POST 06-358
Tang, Wenxing POST 09-429
Tang, Mary POST 10-114
Tangirala, Ramakrishna POST 1076
Taniguchi, Tooru POST 10-107
Tanimoto, Akiko POST 09-422
Tantama, Mathew POST 03-249
Tarekegn, Meron POST 04-322,
POST 04-323
Tasciotti, Ennio POST 06-355
Tashiro, Shinya POST 10-87
Tata, Angela POST 03-311
Tateoka, Chika POST 03-304
Tavernier, Jan POST 05-326
Taylor, Susan S. POST 10-67
Terada, Takaho POST 06-356
Tesmer, John J. POST 10-52
Tesmer, John J. POST 10-55
Textor, Larissa C. POST 03-279
Tezcan, Akif SYMP 09-30
Theobald, Douglas L. POST 08-405
Theodorakis, Emmanuel A. POST
10-121
Thery, Manuel SYMP 08-14
Thomas, Christopher . POST 12150
Thomas, Suzanne POST 03-301
Thulin, Craig D. POST 08-413
Tidor, Bruce POST 12-203
282
Timmerman, Evy POST 05-326
Timucin, Emel POST 12-168
Ting, Alice Y. POST 03-232
Ting, Alice SYMP 07-09
Tischer, Alexander POST 10-103
Titeca, Kevin POST 05-326
Toh, Hiroyuki POST 08-398
Tolbert, Blanton S. POST 10-82
Tomchick, Diana R. POST 03-234
Tomizaki, Kin-ya POST 12-187
Tompa, Peter SYMP 04-06
Toney, Michael POST 12-219
Tong, Liang POST 03-240
Torres, Matthew P. POST 06-351,
POST 08-412, POST 09-432
Totsingan, Filbert POST 12-184
Townsend, Ried POST 06-354
Trans, Denise POST 10-57
Traxlmayr, Michael W. POST 12203
Troemel, Emily POST 05-327
Truscott, Kaye SYMP 08-15
Trylska, Joanna POST 07-379
Tsai, Ching-Ju POST 06-370
Tsai, Shiou-Chaun (Sheryl) POST
03-296
Tseng, Roger POST 11-133
Tsenkova, Roumiana POST 10-80
Tsien, Rodger Y. POST 03-242
Tsien, Roger Y. POST 03-276
Tsubaki, Motonari POST 07-385
Tsuchisaka, Yutaro POST 10-80
Tsui, Vickie POST 04-316
Tsumoto, Kouhei POST 06-358,
10-87, POST 12-154
Tsuruoka, Takaaki POST 12-187
Tu, Ling-Hsien POST 10-59
Turnbull, Joanne L. POST 03-235
U
Udgaonkar, Jayant B. POST 07-378
Uemura, Tatsuya POST 07-382
Uemura, Yasushi POST 03-289
Ueno, Takamasa POST 12-154
Ulusu, Nuray N. POST 03-275
Umemoto, Naoyuki POST 03-245
Uni, Takeshi POST 09-428
Uno, Keisuke POST 08-411
Urbach, Adam POST 03-309
Ushakova, Natalia V. POST 10-64
Usui, Kenji POST 12-187
V
Vadrevu, Ramakrishna POST 12201
Valdes-Garcia, Gilberto POST 07374
Vance, Stephen POST 10-63
Vandebussche, Guy POST 06-349
van Nuland, Nico A. POST 10-78
Vanquickelberghe, Emmy POST
05-326
Varadarajan, Raghavan POST 12165
Varner, Jeffrey D. POST 12-211
Varnes, Philip POST 10-89, POST
10-90
Vazquez Contreras, Edgar POST
07-391
Veeraraghavan, Sudha POST 09431
Veglia, Gianluigi POST 08-414
Venkatesan, Anandakrishnan POST
02-229
Verchere, Bruce POST 10-59
Verhee, Annick POST 05-326
Verschueren, Tim POST 07-371
Vicari, Daniele POST 10-77
Vierling, Elizabeth POST 09-423
Vieux, Ellen POST 04-318
Vinokurov, Konstantin S. POST 03298
Vique, José L. POST 10-98
Voigt, Chris SYMP 09-28
Volker, Mark POST 03-271
Vorotnikova, Elena A. POST 03-298
Voss, John C. POST 06-368
Vu, Nhuan POST 10-113
W
Walden, Patricia M. POST 03-233
Wang, Peter Yingxiao POST 12-145
Wang, Andrew H.-J. POST 03-237
Wang, Lili POST 06-364, POST 11126
Wang, Hui POST 10-59
Wang, Weiping POST 10-69
Wang, Chu POST 03-280
Wang, Ruiying POST 12-217
Wang, Tzu-Chi POST 03-310
Ward, Todd J. POST 04-317
Warner, Lisa R. POST 09-422
Warwicker, Jim POST 12-146
Watson, Jeffrey POST 03-247,
POST 08-403
Weeks, Stephen POST 07-371
Weiner, Zachary POST 11-138
Weininger, Ulrich POST 12-197
Weinstock, Matthew T. POST 12156
Weldon, John E. POST 10-51
Wells, Robert POST 12-176
Wen, Tuan-Nan POST 03-237
Werner, Josephine P. POST 03-265
Wertz, Ingrid POST 04-316
Westwood, Brian POST 05-328,
08-408, POST 08-410
Whelan, Fiona POST 12-169
Wicklow, Donald T. POST 04-317
Wickner, Sue POST 11-135
Wik, Lotta POST 10-109
Williams, Jennie POST 03-231
Williams, Danielle POST 12-157
Williams, Blake POST 12-220
Williamson, James R. POST 07-381
Williamson, James R. POST 10-122
Wilson, Mark A. POST 09-437
Wilson, Ian SYMP 06-43
Winkvist, Maria POST 08-415
Winn, Peter J. POST 12-150
Wismer, Kevin POST 11-137
Wittrup, Karl D. POST 12-203
Woldring, Daniel R. POST 12-182
Woodard, DaNae POST 08-399
Woodruff, Matthew POST 03-294
Woods, Simone POST 03-297
Woolfson, Dek SYMP 09-31
Woolley, G. Andrew POST 12-151
Wu, Chun-Xiang POST 10-87
Wu, Di POST 10-109
Wu, Ruiying POST 10-117
Wu, Xiuifeng POST 12-198
Wyman, Leslie A. POST 03-266
Wysocki, Vicki POST 09-423
X
Xia, Yan POST 12-183
Xiao, Yao POST 09-422
Xin, Li POST 03-238, POST 03258, POST 03-303, POST 10-94
Y
Yabukarski, Filip POST 10-83
Yachnin, Brahm POST 03-250
Yamada, Takuya POST 09-428
Yamada, Aoi POST 12-187
Yamato, Ichiro POST 06-356
Yan, Junhong POST 10-109
Yanaka, Saeko POST 12-154
Yang, Jianyi POST 11-126
Yang, Jin POST 03-242
283
Yang, Heehong POST 06-337
Yang, Weitao POST 07-394
Yang, Meng POST 03-301
Yang, Chen POST 05-331
Yang, Xiaoting POST 10-65
Yang, Na POST 09-438
Yao, Xiaolan POST 03-255
Yasuhara, Kazuma POST 12-202
Yeates, Todd O. POST 03-310,
POST 12-155
Yeates, Todd O. POST 02-225
Yellen, Gary POST 03-249
Yezdimer, Eric M. POST 03-268
Yohda, Masafumi POST 08-398
Yokoyama, Atsuro POST 10-108
Yokoyama, Misaki POST 07-385
Yokoyama, Shigeyuki POST 06-356
Yomo, Tetsuya POST 08-411,
POST 12-162, POST 12-210
Yoshida, Takuya POST 10-107
Yoshimura, Shigehiro POST 07-377
Yousef, Mohammed POST 12-223
Yuan, Xianrui POST 03-268
Yun-hong, Wang POST 03-238,
POST 03-258, POST 10-94
Z
Zanni, Martin T. POST 10-59
Zarzhitsky, Shlomo POST 12-152
Zatsepin , Nadia POST 06-370
Zeman, Jiri POST 04-320
Zhang, Yang POST 11-126
Zhang, Sheng POST 12-153
Zhang, Jun POST 03-238
Zhang, Jinghua POST 10-59
Zhang, Huashan POST 10-69
Zhang, Yu POST 10-114
Zhang, Zhiwen J. POST 12-220
Zhang, Hua POST 06-361
Zhang, Shao-Qing POST 12-202
Zhang, Yuanming POST 11-138
Zhao, Ray POST 04-316
Zhao, Chunxia POST 06-353
Zhao, Cheng POST 09-438
Zhao, Mitchell J. POST 06-362
Zheng, Huimei POST 12-159
Zheng, Jie POST 10-72
Zheng, Yi POST 06-353
Zheng, Zhilli POST 10-91
Zhou, Zijian POST 03-312
Zhou, Li POST 10-122
Zhou, Hu POST 06-364
Zianni, Michael POST 10-96
Ziemba, Brian SYMP 10-33
Zobel, Kerry POST 04-316
284

poster abstracts

Transcription

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